Polyamine conjugates for treatment of infection

ABSTRACT

The present invention relates to methods of preventing or treating an infection or disease caused by an infectious agent. The present invention also relates to the augmentation of the efficacy of existing anti-infective agents by the co-administration of the compounds described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of application Ser. No.08,583,809 filed Jan. 5, 1996, now U.S. Pat. No. 5,744,453, the subjectmatter of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a novel method for treatment of aninfection, especially bacterial or fungal, comprising administering aneffective amount of the disclosed compounds to a patient or host in needthereof.

BACKGROUND OF THE INVENTION

Few developments in the history of medicine have had such a profoundeffect upon human life and society as the development of the power tocontrol infections due to microorganisms.

Although true antibiotics were recognized in folk medicine as long as2500 years ago when the Chinese reported the medicinally beneficialeffects of moldy bean curd, it was not until the nineteenth century whenPasteur founded the science of bacteriology that these substances werestudied systematically. Since that time, the pace of new discoveries hasaccelerated with many new and important antibiotics belonging to variousgroups of compounds being discovered in the nineteenth century.

Although several thousand antibiotics are known, only a relative handfulhave reached the market and achieved commercial importance. Only a veryfew, perhaps 0.3% of the many antibiotics mentioned in the scientificliterature, are now used in medicine and agriculture. A continuing needexists, therefore, for selective and effective antibiotics that do noteasily produce resistance, show an absence of toxicity to the kidney,liver and central nervous system and which are easily administered inoral or parenteral forms. The present invention addresses this need andother needs.

SUMMARY OF THE INVENTION

The present invention relates to a novel method for the treatment orprevention of an infection comprising administering to a host or subjectin need thereof an effective amount of a compound or its saltrepresented by formula (I) ##STR1## in which R₁ can be an H, OH, OR₅,NH₂, NHR₆, or NR₆ R₇ ;

R₂ and R₃ may be the same or different and can be an H, OH, or OR₅ ;

R₄ can be CONH₂, CONHR₆, CONR₆ R₇, CH₂ NH₂, CH₂ NHR₆, CH₂ NR₆ R₇, CO₂--Y--NH₂, CO₂ --Y--NHR₆, or CO₂ --Y--NR₆ R₇ ;

R₅ is a protected or unprotected glycosyl moiety comprising 1-10monosaccharide units in which the glycosidic linkage at the anomericcarbon atom of each monosaccharide unit is independently alpha or beta;

NH₂, NHR₆ and NR₆ R₇ represent an unsubstituted amino group, amonosubstituted amino group and a disubstituted amino group,respectively, in which R₆ and R₇ may be the same or different andrepresent a hydrocarbon group comprising 1-15 carbon atoms substitutedwith one or more unsubstituted, monosubstituted, or disubstituted aminogroups;

Y represents a linear or branched alkylene group comprising 1-10 carbonatoms;

n is an integer from 0-10.

The present inventors have surprisingly and unexpectedly discovered thatthe compounds represented by the formula (I) possess antibiotic activityagainst a wide variety of microorganisms, and may therefore be used, forexample, to prevent or treat bacterial or fungal infections in animals,particularly humans, as well as to serve as disinfectants forsuppressing bacterial or fungal growth, for example, on surfaces such asthose of surgical instruments.

The inventors have also found that the antibiotic compounds useful inthe present method also augment the activity of certain other antibioticcompounds, including conventional ones, such that much less of suchother antibiotic compounds is required to suppress bacterial or fungalgrowth.

Other aspects of the present invention will become apparent to those ofordinary skill in the art upon further consideration of the detaileddescription of the preferred embodiments, presented below, which aremeant to illustrate the basic concepts of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows additional examples of aliphatic amine moieties;

FIG. 2 shows further examples of aromatic amine moieties;

FIG. 3 illustrates the synthetic scheme for the preparation of3α-hydroxy-7-deoxy-12α-(1'α-glucosyl)-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl) amide (may also be referred to as the12-(glycosylated)deoxycholic acid-spermine conjugate, Compound D);

FIG. 4 illustrates the synthetic scheme for the preparation of3α-hydroxy-12-deoxy-7α-(1'α-glucosyl)-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide (may also be referred to as the7-(glycosylated)chenodeoxycholic acid-spermine conjugate, Compound E);

FIG. 5 illustrates the synthetic scheme for the preparation of3α,7α,12α-trihydroxy-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide (may also be referred to as thecholic acid-spermine conjugate, Compound B).

FIG. 6 illustrates the synthetic scheme for the preparation of3α-hydroxy-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide (may also be referred to as thebis(glycosylated)cholic acid-spermine conjugate, Compound C).

FIG. 7 illustrates the synthetic scheme for the preparation of3β-amino-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide trihydrochloride;

FIG. 8A and 8B show the effects of compound H on the MIC of Erythromycinfor E. coli 25922 and P. aeruginosa 27853;

FIG. 9A and 9B show the effects of compound J on the MIC of Erythromycinfor E. coli 25922 and P. aeruginosa 27853; and

FIG. 10A and 10B show the effects of compound N on the MIC ofErythromycin for E. coli 25922 and P. aeruginosa 27853.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is broadly concerned with a novel method fortreatment of an infection comprising administering to a host or subjectin need thereof an effective amount of certain compounds represented byformula (I) ##STR2## in which R₁ can be an H, OH, OR₅, NH₂, NHR₆ or NR₆R₇ ; R₂ and R₃ may be the same or different and can be an H, OH or OR₅ ;R₄ can be CONH₂, CONHR₆, CONR₆ R₇, CH₂ NH₂, CH₂ NHR₆, CH₂ NR₆ R₇, CO₂--Y--NH₂, CO₂ --Y--NHR₆, or CO₂ --Y--NR₆ R₇ ; R₅ is a protected orunprotected glycosyl moiety comprising 1-10 monosaccharide units inwhich the glycosidic linkage at the anomeric carbon atom of eachmonosaccharide unit is independently alpha or beta; NH₂, NHR₆, and NR₆R₇ represent an unsubstituted amino group, monosubstituted amino groups,and a disubstituted amino group, respectively, in which R₆ and R₇ may bethe same or different and represent a linear, branched or cyclichydrocarbon group (e.g., an aliphatic group, a cyclic aliphatic group,an aromatic group or combinations of same) comprising 1-15 carbon atomsoptionally substituted with one or more unsubstituted, monosubstitutedor disubstituted amino groups; Y represents a linear or branchedalkylene group comprising 1-10 carbon atoms; n is an integer from 0-10,preferably 0-3; or its salts.

The degree of substitution of the amino group is determined by thenumber of bonds to hydrogen emanating from the amino group. Thus, anunsubstituted amino group has two N--H bonds (e.g., --CH₂ --CH₂ --NH₂).A monosubstituted amino group has one N--H bond (e.g., --CH₂ --NH--CH₂-- or --CH═NH). A disubstituted amino group has none (e.g., ═CH--NR--CH₂-- or --CH═N--CH═). By "substituted with one or more unsubstituted,monosubstituted or disubstituted amino groups" is meant that thehydrocarbon group comprising 1-15 carbon atoms contains at least oneamino group either within the hydrocarbon backbone (e.g., --CH₂--NH--CH₂ --, --CH₂ --NR--CH₂ --, --CH═N--CH₂, --CH═N--CH═, and thelike) or coming off the backbone (e.g., a primary amine, a secondaryamine, a tertiary amine, an imine or the like, such as --CH₂ --CH₂--NH₂, --CH₂ --CH(--NH₂)--CH₂ --, --CH₂ --CR(NH₂)--CH₂ --, --CH═NH or--CR═NH).

Accordingly, such amino groups are capable of accommodating a charge,for example, in protic media (e.g., --CH₂ --NH₂ .sup.⊕ --CH₂ -- or --CH₂--CH₂ --NH₃.sup.⊕) or on formation of a quaternary ammonium salt (e.g.,--CH₂ --CH₂ --NMe₃.sup.⊕, wherein Me stands for methyl). The preferredcompounds of the present invention include those that are able toaccommodate two or more positive charges. Yet others can accommodatethree, four or even more positive charges.

Additional examples of selected amino group-containing moieties, thatmay be used as R₆ and/or R₇, can be found in FIGS. 1 and 2.

As stated above, the group R₅ can be a protected or unprotected glycosylmoiety, which, in turn, may comprise 1-10 monosaccharide units (e.g., amonosaccharide, a disaccharide, a trisaccharide, etc.). In the presentcase, the term "monosaccharide" is any sugar residue or derivativethereof. The monosaccharide may, for example, be a hexose (e.g.,D-allose, L-allose, D-altrose, L-altrose, D-fucose, L-fucose, D-glucose,L-glucose, D-mannose, L-mannose, D-gulose, L-gulose, D-idose, L-idose,D-galactose, L-galactose, D-rhamnose, L-rhamnose, D-talose, L-talose,and the like, or any deoxy form thereof, e.g., a 2-deoxyhexose, or anyamino-substituted derivative thereof, e.g., an aminosugar, such asD-glucosamine, L-glucosamine, D-galactosamine, L-galactosamine, etc.).Puranoses, deoxyfuranoses, amino-substituted furanoses, and the like arealso suitable, such as D-ribose, L-ribose, D-arabinose, L-arabinose,D-xylose, L-xylose, D-lyxose, L-lyxose, etc.

Furthermore, the protecting groups for the hydroxyl groups (or aminogroups, as the case may be) can be chosen from a wide variety ofprotecting groups appropriate for a given set of conditions. Theseprotecting groups, the choice of which will be apparent to one skilledin the art, may include, but are not limited to, benzyl, pentenyl,pivaloyl, trimethylsilyl, tert-butyldimethylsilyl,tert-butyldiphenylsilyl, triisopropylsilyl, acetyl, tetrahydropyranyl,benzoyl, C₁ -C₃ alkyl, isopropylidene, benzylidene, trifluoroacetyl,(2-methoxyethoxy)methyl, succinyl, orthoester, paramethoxybenzyl, allyl,and the like.

The term "salt", as used herein, denotes acidic and/or basic salts,formed with inorganic or organic acids and/or bases, preferably basicsalts. While pharmaceutically acceptable salts are preferred,particularly when employing the compounds of the invention asmedicaments, other salts find utility, for example, in processing thesecompounds, or where non-medicament-type uses are contemplated. Salts ofthese compounds may be prepared by art-recognized techniques.

Examples of such pharmaceutically acceptable salts include, but are notlimited to, inorganic and organic addition salts, such as hydrochloride,sulphates, nitrates or phosphates and acetates, trifluoroacetates,propionates, succinates, benzoates, citrates, tartrates, fumarates,maleates, methane-sulfonates, isothionates, theophylline acetates,salicylates, respectively, or the like. Lower alkyl quaternary ammoniumsalts and the like are suitable, as well.

In a specific embodiment, the group R₁ has the configuration beta. Inanother, the group R₁ has the configuration alpha. In a particularembodiment, at least one of R₁, R₂, and R₃ represents OH. In anotherembodiment, at least two of R₁, R₂, and R₃ represent OH, and in stillanother embodiment, all three of R₁, R₂, and R₃ represent OH.

The present invention contemplates all other combinations of the variousgroups, including, but not limited to, embodiments in which R₁ and R₂represent OR₅, and R₃ represents OH; R₁ and R₃ represent OR₅, and R₂represents OH; or R₂ and R₃ represent OR₅, and R₁ represents OH.

Furthermore, a method is disclosed wherein R₆ together with the nitrogenatom to which it is attached derives from a polyamine. Suitablepolyamines include, but are not limited to, alkylene diamines, such as1,3-diaminopropane, and 1,12-diaminododecane, and biogenic polyamines(that is, those found in nature), such as 1,4-diaminobutane(putrescine), 1,5-diaminopentane (cadaverine),N-(4-aminobutyl)-1,3-diaminopropane (spermidine, an alkylene triamine),and N-[N-(3-aminopropyl)-4-aminobutyl]-1,3-diaminopropane(spermine, analkylene tetraamine). Other polyamines are also suitable, including butnot limited to, tetraethylenepentamine ("pentamine"),pentaethylenehexamine ("hexamine") and the like, including branchedaliphatic polyamines. With unsymmetrical polyamines, the presentinvention contemplates all other possible points of attachment of thepolyamine to the steroid nucleus. For example, in spermidine, any of thethree amino groups may be attached to the side chain or at the C-3position of the steroid nucleus.

In selected embodiments of the present invention, the group R₁ or R₄ isneither an amino acid nor a peptide.

In especially preferred embodiments of the present invention, thecompound is selected from the group wherein n=2 and,

R₁ =α-OH, R₂ =H, R₃ =OH and R₄ =CO-spermine;

R₁ =α-OH, R₂ =OH, R₃ =OH and R₄ =CO-spermine;

R₁ =α-OH, R₂ =α-D-Glc, R₃ =α-D-Glc and R₄ =CO-spermine;

R₁ =α-OH, R₂ =H, R₃ =α-D-Glc and R₄ =CO-spermine;

R₁ =α-OH, R₂ α-D-Glc, R₃ =H and R₄ =CO-spermine;

R₁ =α-OH, R₂ H, R₃ =OH and R₄ =CO-pentamine;

R₁ =α-OH, R₂ =H, R₃ =OH and R₄ =CO-hexamine;

R₁ =α-OH, R₂ =OH, R₃ =H and R₄ =CO-spermine;

R₁ =α-OH, R₂ =OH, R₃ =H and R₄ =CO-pentamine;

R₁ =α-OH, R₂ =OH, R₃ =OH and R₄ =CO-pentamine;

R₁ =α-OH, R₂ =OH, R₃ =OH and R₄ =CO-hexamine;

R₁ =α-OH, R₂ =α-D-Glc, R₃ =α-D-Glc and R₄ =CO-hexamine;

R₁ =α-OH, R₂ =α-D-Glc, R₃ α-D-Glc and R₄ =CO-pentamine; and

R₁ =α-OH, R₂ =H, R₃ =H and R₄ =CO-hexamine.

Particularly preferred compounds include3α,12α-dihydroxy-7-deoxy-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide (Compound A);3α,7α,12α-trihydroxy-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide (Compound B);3α-hydroxy-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide (Compound C);3α-hydroxy-7-deoxy-12α-(1'α-glucosyl)-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide (Compound D);3α-hydroxy-12-deoxy-7α-(1'α-glucosyl)-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide (Compound E);3α,12α-dihydroxy-7-deoxy-5β-cholan-24-oic acid,N-(3,6,9-triaza-11-aminoundecyl)amide (Compound F);3α,12α-dihydroxy-7-deoxy-5β-cholan-24-oic acid,N-(3,6,9,12-tetraaza-14-aminotetradecyl)amide (Compound G);3α,7α-dihydroxy-12-deoxy-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide (Compound H);3α,7α-dihydroxy-12-deoxy-5β-cholan-24-oic acid,N-(3,6,9-triaza-11-aminoundecyl)amide (Compound I);3α,7α,12α-trihydroxy-5β-cholan-24-oic acid,N-(3,6,9-triaza-11-aminoundecyl)amide (Compound J);3α,7α,12α-trihydroxy-12-deoxy-5β-cholan-24-oic acid,N-(3,6,9,12-tetraaza-14-aminotetradecyl)amide (Compound K);3α-hydroxy-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oic acid,N-(3,6,9,12-tetraaza-14-aminotetradecyl)amide (Compound L);3α-hydroxy-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oic acid,N-(3,6,9-triaza-11-aminoundecyl)amide (Compound M); and3α-hydroxy-7,12-dideoxy-5β-cholan-24-oic acid (or lithocholic acid),N-(3,6,9,12-tetraaza-14-aminotetradecyl)amide (Compound N).

It is of course preferred that the compounds have a degree of puritysuch that they are suitable for use as an anti-infective. Further, thepure or substantially pure compounds are preferably employed in themethods of the present invention. It is understood that one or morecompound(s) of the present invention may be employed in any of themethods described herein.

The compounds represented by formula (I) are useful as anti-infectiveagents, having utility in inhibiting the growth of, including killing,microorganisms. The compounds are particularly useful as broad spectrumantibacterial agents, having activity against both gram-positive andgram-negative bacteria, and as antifungal agents, having activityagainst yeast, mold, or other types of fungi. Thus, the compoundsrepresented by formula (I) may be employed in utilities suitable forsuch antimicrobial or antifungal agents.

The compounds represented by formula (I) may, for example, be used intreating a host infected with a bacterium or fungus, or in preventinginfection of said host by said bacterium or fungus, comprising the stepof administering to the host one or more compounds represented byformula (I) or a pharmaceutically acceptable salt thereof in an amounteffective for prevention or treatment. Treatment of such infectionsaccording to the present invention includes both mitigation as well aselimination thereof.

Hosts administered the compounds represented by formula (I) may beplants or animals, particularly animals such as dogs, cats and otherdomestic mammals and, especially humans. The dosage form and mode ofadministration, as well as the dosage amount, may be selected by one ofordinary skill in the art. The dosage amount will vary with the severityof the infection, and with the size and species of the host. Exemplarydaily dosages for an adult human are those within the range of fromabout 0.001 mg to about 1,000 mg/day, preferably about 0.01 mg to about500 mg/day, most preferably about 0.1 mg to about 200 mg/day. In certaininstances, the preferred ranges may be about 0.5 mg to about 100 mg/day,more preferrably about 1 mg to about 25 mg/day, and most preferably,about 1 mg to about 10 mg/day.

In order to use a compound represented by formula (I) in the method forthe therapeutic treatment of mammals including humans, in particular intreating infection, it is normally formulated in accordance withstandard pharmaceutical practice as a pharmaceutical composition.

The compounds represented by formula (I) may be administered in standardmanner for the disease condition that one desires to treat.Administration to a mammalian host may, for example, be oral, topical,rectal or parenteral. Administration to a plant host may be accomplishedby, for example, application to seed, foliage or other part of theplant, or to the soil. It may also be sprayed over surfaces and over awide area to be treated. For these purposes the compounds of thisinvention may be formulated by means known in the art into the form of,for example, tablets (including lozenges and granules), dragees, pills,ampoules, capsules, aqueous or oily solutions or suspensions, emulsions,dispersible powders, suppositories and sterile injectable aqueous oroily solutions or suspensions. "Pharmaceutical composition" meansphysically discrete coherent portions suitable for medicaladministration. "Pharmaceutical composition in dosage unit form" meansphysically discrete coherent units suitable for medical administration,each containing a daily dose or a multiple (up to four times) or asub-multiple (down to a fortieth) of a daily dose of the active compoundin association with a carrier and/or enclosed within an envelope.Whether the composition contains a daily dose, or for example, a half, athird or a quarter of a daily dose, will depend on whether thepharmaceutical composition is to be administered once or, for example,twice, three times or four times a day, respectively.

Advantageously, the compositions are formulated as dosage units, eachunit being adapted to supply a fixed dose of active ingredients.Tablets, coated tablets, capsules, ampoules and suppositories areexamples of preferred dosage forms according to the invention.

It is only necessary that the active ingredient constitute an effectiveamount, i.e., such that a suitable effective dosage will be consistentwith the dosage form employed in single or multiple unit doses. Theexact individual dosages, as well as daily dosages, are determinedaccording to standard medical principles under the direction of aphysician or veterinarian.

It should be understood that an effective dose of the compound useful inthe present method may vary depending on whether the compound is beingadministered alone or in combination with a second compound. Generally,the effective dose can decrease if the second compound of a combinationalso exhibits anti-infective activity, especially those anti-infectiveagents whose activity is augmented in the presence of the compoundsdisclosed for use in the invention.

The active agents can also be administered as suspensions, solutions andemulsions of the active compound in aqueous or non-aqueous diluents,syrups, granulates or powders. Diluents that can be used inpharmaceutical compositions (e.g., granulates) containing the activecompound adapted to be formed into tablets, dragees, capsules and pillsinclude the following: (a) fillers and extenders, e.g., starch, sugars,mannitol and silicic acid; (b) binding agents, e.g., carboxymethylcellulose and other cellulose derivatives, alginates, gelatine andpolyvinyl pyrrolidone; (c) moisturizing agents, e.g., glycerol; (d)disintegrating agents, e.g., agar-agar, calcium carbonate and sodiumbicarbonate; (e) agents for retarding dissolution, e.g., paraffin; (f)resorption accelerators, e.g., quaternary ammonium compounds; (g)surface active agents, e.g., cetyl alcohol, glycerol monostearate; (h)adsorptive carriers, e.g., kaolin and bentonite; (i) lubricants, e.g.,talc, calcium and magnesium stearate and solid polyethylene glycols.

The tablets, dragees, capsules and pills comprising the active agent canhave the customary coatings, envelopes and protective matrices, whichmay contain opacifiers. They can be so constituted that they release theactive ingredient only or preferably in a particular part of theintestinal tract, possibly over a period of time. The coatings,envelopes and protective matrices may be made, for example, frompolymeric substances or waxes.

The active ingredient can also be made up in microencapsulated formtogether, with one or several of the above-mentioned diluents.

The diluents to be used in pharmaceutical compositions adapted to beformed into suppositories can, for example, be the usual water-solublediluents, such as polyethylene glycols and fats (e.g., cocoa oil andhigh esters, (e.g., C₁₄ -alcohol with C₁₆ -fatty acid]) or mixtures ofthese diluents.

The pharmaceutical compositions which are solutions and emulsions can,for example, contain the customary diluents (with, of course, theabove-mentioned exclusion of solvents having a molecular weight below200, in the presence of a surface-active agent), such as diluents,dissolving agents and emulsifiers. Specific non-limiting examples ofsuch diluents are water, ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (for example,ground nut oil), glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitol or mixtures thereof.

For parenteral administration, solutions and suspensions should besterile, e.g., water or arachis oil contained in ampoules and, ifappropriate, blood-isotonic.

The pharmaceutical compositions which are suspensions can contain theusual diluents, such as liquid diluents, e.g., water, ethyl alcohol,propylene glycol, surface active agents (e.g., ethoxylated isostearylalcohols, polyoxyethylene sorbitols and sorbitan esters),polycrystalline cellulose, aluminum methahydroxide, agar-agar andtragacanth, or mixtures thereof.

The pharmaceutical compositions can also contain bulking agents andpreservatives, as well as perfumes and flavoring additions (e.g.,peppermint oil and eucalyptus oil), and sweetening agents, (e.g.,saccharin and aspartame).

The pharmaceutical compositions will generally contain from about 0.0001to 90 wt. %, preferably about 0.01 to 10 wt. % of the active ingredientby weight of the total composition. In addition to the active agent, thepharmaceutical compositions and medicaments can also contain otherpharmaceutically active compounds.

Any diluent in the pharmaceutical compositions of the present inventionmay be any of those mentioned above in relation to the pharmaceuticalcompositions. Such compositions may include solvents of varyingmolecular weight as the sole diluent.

The active compound is administered perorally, parenterally (forexample, intramuscularly, intraperitoneally, subcutaneously,transdermally or intravenously), rectally or locally, preferably orallyor parenterally, especially perlingually, or intravenously.

The dosage rate, e.g., 0.001 to 200 mg/kg of body weight, will be afunction of the nature and body weight of the human or animal subject tobe treated, the individual reaction of this subject to the treatment,type of formulation in which the active ingredient is administered, themode in which the administration is carried out and the point in theprogress of the infection or interval at which it is to be administered.Thus, it may in some case suffice to use less than a minimum dosagerate, while other cases an upper limit must be exceeded to achieve thedesired results. Where larger amounts are administered, it may beadvisable to divide these into several individual administrations overthe course of the day.

In addition to the compounds of the present invention, thepharmaceutical composition of this invention may also contain, or beco-administered with, one or more known drugs selected from otherclinically useful anti-infective agents. Examples of such anti-infectiveagents include, for example, amikacin, bacitracin, candicidin,capreomycin, cephalosporins (cefazolin, cephaloglycine, cephaloridine,cephalothin, cephapirin sodium, cephradine), chloramphenicol, colistin(polymyxin), cycloserine, dactinomycin, erythromycin, fusidic acid,gentamicin, gramicidin, kanamycin, lincomycins (clindamycin,lincomycin), neomycin, oleandomycins (oleandomycin, troleandomycin),paromomycin, penicillins (amoxicillin, ampicillin, carbenicillin,carbenicillin, indanyl ester, cloxacillin, dicloxacillin, hetacillin,methacillin, nafcillin, oxacillin, penicillin G (benzylpenicillin),penicillin V (phenoxymetholpenicillin), phenethicillin), rifampin,spectinomycin, staphylomycin, streptomycins (dihydrostreptomycin,streptomycin), tetracyclines (chlortetracycline, demeclocycline,deoxycycline, methacycline, minocycline, oxytetracycline, tetracycline),tyrothricin, vancomycin, and viomycin. Preferred anti-infective agentsare those that exhibit augmented or enhanced activity in the presence ofthe compounds of the present invention, such as erythromycin.

The anti-infective compounds provide action against specific organismssusceptible to them. Examples of microorganisms that the compoundsrepresented by formula (I) are believed to be active against include,but are not limited to alpha-streptococci, beta-streptococci,Diplococcus pneumoniae, Staphylococcus species, Bacillus anthracis,clostridia spp., Corynebacterium xerose, Haemophilus ducreyi,Haemophilus influenzae, Escherichia coli, Klebsiella-Enterococcusspecies, Neisseria species, Proteus mirabilis, Salmonella typhosa,Pseudomonas aeruginosa, Histoplasma capsulatum, Coccidioides immitis,Candida species, Blastomyces dermatitidis, Rhondototorula, Cryptococcusneoformans, Sporothrix schenckii, Mucor mucedo and Aspergillusfumigatus.

Examples of infections, which may respond to treatment or which may beprevented by administration of the compounds represented by formula (I)include, but are not limited to, skin and soft tissue infections,genitourinary-tract infections, gastrointestinal infections, gonorrhea,respiratory infections, meningitis, aspergillosis, cryptococcosis(torulosis), North American blastomycosis, systemic candidiasis,coccidioidomycosis, histoplasmosis, zygomycosis and subacute bacterialendocarditis.

The appropriate solid or liquid vehicle or diluent may be selected, andthe compositions prepared, by methods known to one of ordinary skill inthe art. Prevention or treatment of simultaneous infections by more thanone bacterium or fungus, or combinations thereof is, of course,contemplated.

The compounds represented by formula (I) may also be employed asantimicrobial agents useful in inhibiting the growth of, includingkilling, microorganisms present on a surface or in a medium outside aliving host. The present invention therefore provides a method forinhibiting the growth of at least one bacterium or fungus present on asurface or in a medium, comprising the step of contacting the surface ormedium with one or more compounds represented by formula (I), or a saltthereof, in an amount effective for the inhibition. Thus, the inventivecompounds may be employed, for example, as disinfectants for surfacetreatments, such as disinfection of surgical instruments, or aspreservatives for a variety of solid and liquid media susceptible tomicrobial growth. Suitable amounts of the compounds may be determined bymethods known to one of ordinary skill in the art. Compositionscomprising at least one compound represented by formula (I), or a saltthereof in an amount effective for inhibiting the growth of at least onebacterium or fungus, and a vehicle or diluent, are also provided by thepresent invention.

The following examples further illustrate the invention, and are notintended to in any way limit the present claims.

EXAMPLES

1. Synthesis of 3β-Amino-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oic Acid,Methyl Ester

1.1. Preparation of 2,3,4,6-Tetra-O-benzyl-α-D-glucopyranose

Methyl-α-D-glucopyranose (100 g, 0.516 mol) is suspended in benzylchloride (400 mL, 3.5 mol) with KOH pellets (336 g, 6 mol), and themixture is stirred using a mechanical stirrer at 120-130° C. for 3 h.The reaction mixture is cooled and water (800 mL) is added to dissolvethe crystalline mass, which is extracted with ether (2×200 mL). Thecombined organic layer is washed with water (2×500 mL) and dried (Na₂SO₄). The solvents are removed by vacuum distillation to give the crudemethyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranoside for the next reaction.

To a stirred solution of above crude compound in glacial acetic acid(700 mL) at 110° C. is added 3N sulfuric acid (120 mL) dropwise during15 min. After 3 h the reaction mixture is cooled to room temperature andleft over night for crystallization of product. The crystals arefiltered, washed consecutively with water (4×500 mL) and methanol (2×250mL), and air dried to afford 2,3,4,6-tetra-O-benzyl-α-D-glucopyranose(115 g, 41% overall two steps) as a white powder (mp 150-51° C., lit.151-152° C.; See, Perrine, T. D. et al. J. Org. Chem. (1967) 32:664).TLC (EtOAC:Hexane 3:7) R_(f) 0.2. IR (KBr): 3362, 3030, 2911, 2863,1454, 1357, 1146, 1088 cm⁻¹. ¹ H NMR (300 MHz, CDCl₃): δ 7.38-7.10 (m,20H), 5.21 (d, J=3.3 Hz, 1H), 4.98-4.44 (m, 9H), 4.25 (m, 1H), 3.72-3.50(m, 4H). Anal. Calc. for C₃₄ H₃₆ O₆ : C, 75.53; H, 6.71. Found: C,75.68; H, 6.80.

1.2. Preparation of Phenyl2,3,4,6-Tetra-O-benzyl-1-thio-D-glucopyranoside

To a stirred solution of 2,3,4,6-tetra-O-benzyl-α-D-glycopyranose (108g, 0.2 mol) and phenyl disulfide (53 g, 0.24 mol) in dichloromethane(500 mL) is added tri-n-butylphosphine (60 mL, 90%, 0.22 mol). Afterallowing the reaction mixture to stir at room temperature for 15 h, itis poured into a solution of saturated aqueous sodium bicarbonate (600mL) and stirred for 10 min. The organic layer is separated, washed withwater (2×500 mL), dried (Na₂ SO₄) and concentrated. The oily residue isdissolved in hexane (500 mL) and chilled to 0° C. to give phenyl2,3,4,6-tetra-O-benzyl-1-thio-D-glucopyranoside (75 g, 60%) as a whitesolid (mp 85-86° C., lit. 84-85° C. for β-thio compound; See, Ferrier,R. J. et al. Carbohyd. Res. (1973) 27:55). TLC (EtOAC:Hexane 1:3) R_(f)0.6. IR (KBr): 3061, 3030, 2900, 2865, 1584, 1494, 1453, 1358, 1125,1085, 1070, 1029 cm⁻¹. ¹ H NMR (300 MHz, CDCl₃): δ 7.70-7.00 (m, 25H),4.90-4.40 (m, 9H), 3.80-3.40 (m, 6H). Anal. Calc. for C₄₀ H₄₀ O₅ S: C,75.92; H, 6.38, S, 5.06. Found: C, 75.99; H, 6.39; S, 5.12.

1.3. Preparation of Phenyl2,3,4,6-Tetra-O-benzyl-1-thio-D-glucopyranoside S-Oxide

To a stirred cooled (-78° C.) solution of phenyl2,3,4,5-tetra-O-benzyl-1-thio-D-glucopyranoside (130 g, 0.2 mol) indichloromethane (400 mL) is added dropwise over a period of 20 min asolution of mCPBA (74%, 58.31 g, 0.25 mol) in dichloromethane (300 mL)The mixture is stirred and allowed to warm up to -30° C. The mixture isthen filtered. The filtrate is washed with saturated aqueous sodiumbisulfite (2×300 mL), sodium bicarbonate (2×400 mL), brine (400 mL) andwater (2×400 mL). The organic layer is dried (Na₂ SO₄) and concentrated.Flash chromatography (CH₂ Cl₂ :EtOAC 9:1) of the residue furnishes theabove-referenced sulfoxide mixture (127 g, 95%) as a white solid (mp120-122° C.) TLC (EtOH:CH₂ Cl₂ 1:9) R_(f) 0.3. IR (KBr): 3060, 3030,2910, 2867, 1495, 1450, 1360,1210, 1136, 1092, 1049 cm⁻¹. ¹ H NMR(CDCl₃): δ 7.72-7.14 (m, 25H), 5.12-4.42 (m, 9H), 4.40-3.30 (m, 6H).Anal. Calc. for C₄₀ H₄₀ O₆ S: C, 74.04; H, 6.22; S, 4.93. Found: C,74.10; H, 6.26; S, 4.99.

1.4. Preparation of 3α-p-Methoxybenzoate-5β-cholan-24-oic Acid, MethylEster

To a solution of methyl cholate (42.2 g, 0.1 mol), p-anisoyl chloride(20 mL, 0.133 mol) and DMAP (1 g) in pyridine (500 mL) is stirred andrefluxed for 8 h. Additional p-anisoyl chloride (10 mL, 0.67 mol) isadded and stirred 12 h. The reaction mixture is concentrated, and theresidue is dissolved in dichloromethane (600 mL). The solution is washedconsecutively with 1N HCl (2×500 mL) and water (3×500 mL), dried (Na₂SO₄) and the solvent allowed to evaporate. Crystallization of theresidue from EtOAC/hexane (1:1) furnishes the desired acid ester (40 g,72%) as a white solid (mp 179-180° C.). TLC (EtOAC:Hexane 7:3) R_(f)0.7.

1.5. Preparation of3α-p-Methoxybenzoate-7α,12α-di(2',3',4',6'-tetra-O-benzyl-1'α-glucosyl)-5β-cholan-24-oicAcid, Methyl Ester

Triflic anhydride (30 mL, 0.178 mol) is added to cooled toluene (300 mL,-78° C.) and stirred for 5 min. To this solution, the dried (byazeotropic distillation from toluene) sulfoxide from 1.3 (97 g, 0.1495mol) dissolved in toluene (300 mL) is added dropwise. After 15 min ofstirring, a solution of dried (by azeotropic distillation with toluene)2,6-di-ter-butyl-4-methyl-pyridine (30.8 g, 0.150 mol) in toluene (100mL) is added to the reaction mixture and stirred for 10 min at -78° C.To this reaction mixture, dried (by azeotropic distillation withtoluene) acid ester from 1.4 (33.36 g, 0.06 mol) in CH₂ Cl₂ and toluene(1:1, 200 mL) is added dropwise. The reaction progress is monitored byTLC. The temperature of the reaction mixture is slowly brought to -50°C. (during 45 min) and during this time the spot of acid ester from 1.4on the TLC disappeared completely. The reaction mixture is poured into asaturated aqueous solution of sodium bicarbonate (1000 mL) and stirredfor 10 min. The organic layer is separated, and the aqueous layer isextracted with dichloromethane (2×100 mL). The combined organic layersis washed with water (3×500 mL), dried (Na₂ SO₄) and concentrated. Theresidue purified by flash chromatography (EtOAC:Hexane=1:9 to 1:4) tofurnish the desired bis(glycosylated) acid ester (84 g, 87%) as a whitefoam (mp 46-48° C.). TLC (EtOAC:Hexane 1:3) R_(f) 0.3. IR (KBr): 3084,3062, 3028, 2936, 2867, 1735, 1707, 1605, 1496, 1453, 1360,1321,1275,1254,1210, 1165, 1097, 1073, 1030 cm⁻¹. ¹ H NMR (CDCl₃): δ7.60-6.70 (m, 43H), 5.95 (d, 1H, J=9 Hz), 4.99 (d, 1H, J=3.6 Hz), 4.93(d, 1H, J=6 Hz), 4.88-3.29 (m, 31H), 2.68-0.65 (m, 37H). Fab MS: 1624(M+Na)⁺. Anal. Calc. for C₁₀₁ H₁₁₆ O₁₇ : C, 75.71; H, 7.30. Found, C,75.59; H, 7.31.

1.6. Preparation of7α,12α-Di(2',3',4',6'-tetra-O-benzyl-1'α-glucosyl)-5.beta.-cholan-24-oicAcid

To a stirred solution of the product from 1.5 (24 g, 15 mmol) in THF(150 mL), NaOH (10 g, 250 mmol) in 95% Ethanol (200 mL) is added andrefluxed for 48 h. The reaction mixture is then concentrated, and theresidue is dissolved in ethyl acetate (300 mL), washed with water (2×250mL), saturated aqueous sodium bicarbonate (2×300 mL), brine (300 mL) anddried (Na₂ SO₄). Solvent is evaporated and the resulting desiredcompound (18.5 g, 85%) is used for the next step without furtherpurification. TLC (EtOAC:Hexane 1:3) R_(f) 0.4.

1.7. Preparation of7α,12α-Di(2',3',4',6'-tetra-O-benzyl-1'α-glucosyl)-5.beta.-cholan-24-oicAcid, Methyl Ester

A cooled (-10° C.) solution of diazomethane in ether (100 mL, generatedfrom 5.35 g of diazalid, 25 mmol) is added to a cooled (-10° C.)solution of the product from 1.6 (18.5 g, 12.74 mmol) in ether (100 mL).After 1 h, excess diazomethane is destroyed by adding glacial aceticacid (2 mL). The reaction mixture is washed consecutively with saturatedaqueous sodium bicarbonate (2×400 mL), brine (300 mL), and water (300mL), dried (Na₂ SO₄) and concentrated. The residue is purified by flashchromatography (EtOAC:Hexane 3:17) to furnish the desired ester (13 g,70%) as a gum. TLC (EtOAC:Hexane 1:3) R_(f) 0.6. IR (Neat): 3450, 2925,2866, 1736, 1453, 1362, 1158, 1071, 1030 cm⁻¹. ¹ H NMR (CDCl₃): δ7.40-6.50 (m, 40H), 5.10-3.40 (m, 33H), 2.40-0.71 (m, 38H). Anal. Calc.for C₉₃ H₁₁₀ O₁₅ : C, 76.08; H, 7.56. Found: C, 74.79; H, 7.50.

1.8. Preparation of3β-Azido-7α,12'-di(2',3',4',6α-tetra-O-benzyl-1'α-glucosyl)-5β-cholan-24-oicAcid, Methyl Ester

To a cooled (0° C.) solution of methyl bis(glucosyl)cholate from 1.7 (13g, 8.87 mmol) and pyridine (2.5 mL, 31 mmol) in dichloromethane (50 mL),triflic anhydride is added and allowed to stir for 20 min. To thismixture, a solution of sodium azide (2.6 g, 40 mmol) in DMF/DMPU (1:1,250 mL) is then added at -20° C. The reaction mixture is allowed to warmup to room temperature, where it is stirred overnight. The solvents areevaporated, and the residue is dissolved in dichloromethane (200 mL),washed with water (3×200 mL), dried (Na₂ SO₄), and concentrated. Flashchromatography of the residue on silica (EtOAC:Hexane 3:17) furnished 10g (75%) of azide compound as a white solid (mp 112-114° C.). TLC(EtOAC:Hexane 1:4) R_(f) 0.6. IR (KBr): 3085, 3061, 3029, 2921, 2867,2097, 1735, 1603, 1495, 1452, 1360,1256,1207, 1160, 1091, 1071, 1031cm⁻¹. ¹ H NMR (CDCl₃): δ 7.37-6.84 (m, 40H), 5.15 (d, 1H, J=4 Hz), 4.95(d, 1H, J=4 Hz), 4.86-4.26 (m, 15H), 4.08-3.40 (m, 16H), 3.62 (s, 3H),2.60-0.71 (m, 37H), 1.02 (d, 3H), 0.89 (s, 3H) and 0.63 (s, 3H). Fab MS:1515 (M+Na)⁺. Anal. Calc. for C₉₃ H₁₁₀ O₁₄ N₃ : C, 74.76; H, 7.43; N,2.81. Found: C, 74.84; H, 7.40; N, 2.79.

1.9. Preparation of3β-Amino-7α,12α-di(2',3',4',6'-tetra-O-benzyl-1'α-glucosyl)-5β-cholan-24-oicAcid, Methyl Ester

A solution of compound azide of 1.8 (11 g, 7.38 mmol) and Ph₃ P (5.76 g,22 mmol) in 90% aqueous THF (100 mL) is stirred and refluxed for 48 h.The reaction mixture is concentrated, and the residue is purified byflash chromatograph (CH₂ Cl₂ and then CH₂ Cl₂ :EtOH=98:2 to 9:1) to givethe desired 3-amino compound (6 g, 56%) as a white solid (mp 43-45° C.).TLC (EtOH:CH₂ Cl₂ 1:19) R_(f) 0.15. IR (KBr): 3418, 2922, 2868, 1736,1496, 1453, 1362, 1161, 1071, 1032 cm⁻¹. ¹ H NMR (CDCl₃): δ 7.38-6.84(m, 40H), 5.10-3.48 (m, 33H), 2.62-0.70 (m, 37H). Anal. Calc. for C₉₃H₁₁₂ O₁₄ N: C, 76.08; H, 7.70; N, 0.95. Found: C, 75.82; H, 7.71; N.0.89.

1.10. Preparation of 3β-Amino-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oicAcid, Methyl Ester

To a solution of the 3-amino compound of 1.9 (14.65 g, 10 mmol) intoluene (50 mL) and ethanol (200 mL) is added formic acid (15 mL) andpalladium hydroxide (20%) on carbon (15 g). The resulting mixture isstirred for 24 h under a hydrogen atmosphere at 40 psi. TLC indicatedincomplete hydrogenolysis. Additional formic acid (4 mL) and catalyst (4g) is then added, and the hydrogenation reaction allowed to proceed foranother 24 h. The reaction mixture is then filtered through sand over amembrane filter and concentrated. The filtrate is then mixed with ethylacetate to form a precipitate. (In some instances, the methanol solventfrom the hydrogenation reaction may need to be removed.) The filteredprecipitate is then dissolved in 25 mL deionized water and freeze-dried.Flash Chromatography gave 2.82 g (38%) of the deprotected amino cholateester as white foam (mp 170-172° C., decomp.). TLC (MeOH:CH₂ Cl₂:Isopropylamine 2:2:1) R_(f) 0.15. IR (KBr): 3450, 2932, 1736, 1595,1451, 1381, 1151, 1023 cm⁻¹. ¹ H NMR (CDCl₃): δ 5.05 (d, 1H), 4.80 (d,1H), 3.91-3.10 (m, 15H), 2.50-0.58 (m, 37H). MS (Fab): 746 (M+H)⁺. Anal.Calc. for C₃₇ H₆₃ O₁₄ N: C, 59.56; H, 8.52; N, 1.88. Found: C, 54.60; H,8.47; N, 2.49.

The corresponding 3α-amino compound can be obtained from the 3β-hydroxystarting material similarly. The 3β-hydroxy starting material can beobtained, for example, by treatment of methyl cholate with diethylazidodicarboxylate in the presence of formic acid and triphenylphosphine with inversion of stereochemistry to provide the methyl3β-O-formylcholate, which, subsequently, can be hydrolyzed ormanipulated, as needed.

2. Preparation of3α-p-Methoxybenzoate-7α,12α-di(1'α-glucosyl)-5.beta.-cholan-24-oic Acid,Methyl Ester

To a solution of the acid ester of 1.5 (10 mmol; see, above) in toluene(50 mL) and ethanol (200 mL) is added formic acid (15 mL) and palladiumhydroxide (20%) on carbon (15 g). The resulting mixture is stirred for24 h under a hydrogen atmosphere at 40 psi. (Additional formic acid andcatalyst can be added, if desired, if TLC analysis reveals that thereaction is incomplete after the initial 24 h reaction period. A second24 h reaction period can then be initiated.) The reaction mixture isthen filtered through sand over a membrane filter and concentrated. Thefiltrate is then mixed with ethyl acetate to form a precipitate. (Someof the methanol solvent from the hydrogenation reaction may need to beremoved.) The filtered precipitate is then dissolved in 25 mL deionizedwater and freeze-dried. Subjecting the residue to flash columnchromatography gave the title compound in ca. 38% yield.

¹ H NMR (CD₃ OD): δ 0.71 (s, 3H, 18-H), 0.90 (d, 3H, 21-H, J=6.6 Hz),0.93 (s, 3H, 19-H), 1.0-2.6 (m), 3.2-3.4 (m, 2H), 3.55 (s, 3H, CO₂ CH₃),3.65 (m), 3.76 (s, 3H, anisoyl-4-methyl), 4.83 (d, 1H, anomeric H), 5.02(d, 1H, anomeric H), 6.87 (d, 2H, anisoyl aromatic, J=9 Hz), 7.92 (d,2H, anisoyl aromatic, J=9 Hz).

3. Synthesis of the Activated Ester of Deoxycholate

Triethylamine (10 mL, 71.2 mmol) is added to a stirred solution of thesodium salt of deoxycholic acid (15 g, 34.7 mmol), N-hydroxysuccinimide(7.5 g, 65.2 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide(13.2 g, 69.3 mmol, EDC) in dichloromethane. The mixture is stirred for12 h. The reaction mixture is then diluted with water (150 mL) andextracted twice with dichloromethane. The organic layers are combined,dried over MgSO₄, filtered, and concentrated under reduced pressure toprovide a solid residue. The residue is recrystallized from ethylacetate-petroleum ether to give 5.5 g (30%) of product. Selected ¹ Hresonances: (270 MHz, CDCl₃): δ 4.00 (br s, 1H, C12), 3.6 (m, 1H, C3),1.03 (d, 3H, C21), 0.9 and 0.68 (s, 3H each, angular methyls ofsteroid).

4. Synthesis of the Deoxycholic Acid-Spermine Conjugate (Comp. A ofTable 1)

Spermine (0.3 g, 1.18 mmol) is added to a stirred solution of theactivated ester of deoxycholate from Example 3 (0.15 g, 0.28 mmol) andtriethylamine (0.1 mL, 0.71 mmol) in dichloromethane. The mixture isstirred for 0.5 h and a precipitate is observed. The solids are filteredthrough a buchner funnel. The filtrate is washed with water (10 mL). Theorganic layer is concentrated to give a residue (0.18 g). The residue isacidified with methanolic trifluoroacetic acid. The resulting solutionis purified by reverse phase chromatography to give 0.14 9 (80%) of thesteroid-polyamine conjugate. Selected ¹ H resonances: (270 MHz, CD₃ OD):δ 3.98 (br s, 1H, C12), 3.55 (m, 1H, C3), 3.4 (br t, 2H, sperminemethylenes next to amide linkage), 3.0 (br s, 10H, spermine methylenesexcept those next to amide), 1.03 (d, 3H, C21), 0.9 and 0.68 (s, 3Heach, angular methyls of steroid). High resolution mass spectrometryconfirmed the proper molecular weight.

In the same fashion, other non-glycosylated amphiphatic steroidalcompounds, including but not limited to cholic acid or chenodeoxycholicacid, may be conjugated to a polyamine molecule, including but notlimited to ethylene diamine, diethylene triamine, spermidine, otherpolyalkylenepolyamines, and the like.

5. Synthesis of 3α-Hydroxy-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oic Acid

To a stirred solution of the methylcholate product of Example 2, above,(15 mmol) in THF (150 mL) is added NaOH (10 g, 250 mmol) in 95% ethanol(200 mL). The reaction mixture is refluxed for 48 h. The reactionmixture is then concentrated, acidified with dilute Hcl and the residueis dissolved in ethyl acetate (300 mL), washed with water (2×250 mL),saturated aqueous sodium bicarbonate (2×300 mL), brine (300 mL) anddried (Na₂ SO₄). Solvent is evaporated to provide the7,12-bis-α-perbenzylglucosylcholic acid product in 80% yield. (See, FIG.6.) Activation of the carboxylic acid group is carried out as follows.

6. Synthesis of the Bis(glycosylated)cholic Acid-Spermine Conjugate(Comp. C of Table 1) via the Activated Acid

Triethylamine (120 μL, 0.8 mmol) is added to a stirred solution of thecholic acid product of Example 5 (0.3 g, 0.2 mmol), N-hydroxysuccinimide(72 mg, 0.6 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (160mg, 0.8 mmol) in dichloromethane. The mixture is stirred for 12 h. Afterthis time, the reaction mixture is diluted with water (50 mL) andextracted twice with dichloromethane. The organic layers are combined,dried over MgSO₄, filtered, and concentrated under reduced pressure toprovide a solid residue 0.33 g (96%) of the activated ester. (See, Comp.5 of FIG. 6.)

To a stirred solution of the activated ester (0.15 g, 0.089 mmol) andtriethylamine (50 mL, 0.35 mmol) in dichloromethane is added spermine(0.3 g, 0.61 mmol). The mixture is stirred for 0.5 h and a precipitateis observed. The solids are filtered over a buchner funnel. The filtrateis washed with water (10 mL). The organic layer is concentrated to givea residue (0.18 g). The residue is acidified with methanolictrifluoroacetic acid. The resulting solution is purified by reversephase chromatography to give 0.14 g (85%) of the protectedbis(glycosylated)cholic acid-spermine conjugate.

In the same fashion, other glycosylated amphiphatic steroidal compounds,including but not limited to the mono-, di-, or triglycosylated forms(as appropriate) of cholic acid, 7-deoxycholic acid, or chenodeoxycholicacid, may be conjugated to a polyamine molecule, including but notlimited to ethylene diamine, diethylene triamine, spermine, spermidine,other polyalkylenepolyamines, and the like.

7. Deprotection of the Protected Bis(glycosylated)cholic Acid-SpermineConjugate

A hydrogenation flask is charged with a solution of the protectedbis(glycosylated)cholic acid-spermine conjugate (0.11 g, 0.06 mmol; see,above) in a mixture of methanol (20 mL) and benzene (4 mL) or THF,followed by Pd(OH)₂ catalyst and formic acid (1 mL) or hydrochloricacid. The reaction mixture is shaken under a hydrogen atmosphere at 50psi for 40 h. The catalyst is filtered off with Celite®, and the solventis removed by evaporation under reduced pressure. The product ispurified over Sephadex-LH-20 gel, eluting with MeOH, to give the desiredbisglycosteroid-spermine conjugate (Comp. C).

8. Synthesis of the 12α-(O-Glucosyl)deoxycholic Acid-Spermine Conjugate(Comp. D of Table 1 and Comp. 6 of FIG. 3)

8.1. Preparation of 3α-O-CBZ-Deoxycholic Acid, Methyl Ester (Comp. 1 ofFIG. 3)

A mixture of methyldeoxycholate (25 g, 61 mmol), benzylchloroformate(17.0 g, 14 mL, 100 mmol), dimethylaminopyridine (1.22 g, 10 mmol),pyridine (30 mL) and dioxane (150 mL) are stirred at room temperature3h, the additional amounts of the benzylchloroformate (12.0 g, 10 mL)are added two times in 2 h to complete reaction. Total amount of thebenzylchloroformate is 41.0 g (34 mL). The reaction mixture is pouredinto a separatory funnel, water (500 mL) and ethyl acetate (300 mL) areadded. The organic layer is washed with water (500 mL×2), dried oversodium sulfate, concentrated to give an oil. The product is purified onflash chromatography over silica gel (EA-Hexane 1:1) providing 24.0 g(73% yield) of comp. 1 as a thick oil. TLC (EA:Hexane 2:5) R_(f) 0.65.IR (neat): 3553 (OH), 2943, 2869 (CH), 1742 (C═O), 1453, 1389,1263(arom.), 944, 911, 789, 747, 696 cm⁻¹. ¹ H NMR (CDCl₃): δ 7.38 (s, 5H),5.15 (s, 2H), 3.6 (s, 3H), 2.0-1.0 (m, 24H), 0.96 (d, 3H, J=6 Hz), 0.86(s, 3H), 0.65 (s, 3H).

8.2. Preparation of 3α-O-CBZ-12α-(Tetra-O-benzyl-O-glucosyl)deoxycholicAcid, Methyl Ester (Comp. 2 of FIG. 3)

Triflic anhydride (2.08 g, 1.26 mL, 7.4 mmol) is added to dry toluene(100 mL), chilled to -75° C. with acetone-dry ice bath, thenphenylsulphenyl tetra-O-benzylglucopyranoside (glucosulfoxide) (5.06 g,7.4 mmol) is added dropwise, and in 10 minutes the2,6-tert-butyl-4-methyl-pyridine, and then 3-O-CBZ-Deoxymethyl cholate 1is added dropwise. When TLC showed the reaction is finished, it isquenched by sodium bicarbonate (saturated solution, 200 mL) at -25 to-30° C. The organic layer is dried over sodium sulfate, concentrated invacuum at +50 to +60° C. The residue on flash chromatography (EA-Hexane,20% of EA) afforded 2 (1.8 g, 29%), as thick colorless oil. TLC(EA-Hexane 2:5) R_(f) 0.70. ¹ H NMR(CDCl₃): δ 7.3 (m, 24H), 4.4-5.0 (m,10H), 3.6 (s, 3H), 3.4-4.0 (m, 7H), 1.0-1.95 (m, 40H), 0.92 (d, 3H),0.82 (s, 3H), 0.56 (s, 3H).

8.3. Preparation of 12α-(O-Glucosyl)deoxycholic Acid, Methyl Ester(Comp. 3 of FIG. 3)

The comp. 2 (1.6 g, 1.47 mmol) is dissolved in ethyl acetate (15 mL) andethanol (50 mL) together with catalyst Pd(OH)₂ /C (500 mg). Using a Parrshaker, the reaction mixture is pressurized under hydrogen at 50 psi for24 h. The catalyst is filtered off, and the filtrate is evaporated togive a crystalline residue. The residue is purified by flashchromatography (EtOH-DCM 2:8) to afford comp. 3 (0.65 g, yield 72%) aswhite crystals, m.p. 186-188° C. TLC (EtOH-DCM 2:8) R_(f) 0.5. IR(neat): 3510, 2943, 2585, 1690, 1452, 1376, 1148, 1090, 1050 cm⁻¹. ¹ HNMR: δ 5.05 (d, 1H, J=3 Hz), 3.9 (s, 1H), 3.7-3.8 (m, 3H), 3.6 (s, 3H),2.2-1.4 (m, 40H), 0.95 (d, 3H), 0.90 (s, 3H), 0.72 (s, 3H).

8.4. Preparation of 12α-(O-Glucosyl)deoxycholic Acid, Hydrazide (Comp. 4of FIG. 3)

The methyl ester 3 (0.6 g, 1.1 mmol) is refluxed in 5 mL ofEtOH-hydrazine hydrate (10:1) for 3 h. The solvent is evaporated, water(50 mL) added, then distilled off to remove excess of hydrazine hydrate.The residue is azeotroped with toluene to afford a colorless crystallinehydrazide 4 (0.50 g, yield 81%, m.p. 180-182° C.). TLC (EtOH-DCM 2:5)R_(f) 0.15. Anal. Calc. for C₃₀ H₅₂ N₂ O₈ : N 5.0. Found: N 4.81. IR(KBr) 3393, 2907, 2863, 1633, 1543, 1452, 1372, 1144, 1016, 704 cm⁻¹.

8.5. Preparation of 12α-(O-Glucosyl)deoxycholic Acid, Azide (Comp. 5 ofFIG. 3)

Hydrazide 4 (0.5 g, 0.88 mmol) is dissolved in 5 mL of 10% HCl at +1 to+3° C. to give a clear solution. Then NaNO₂ (0.14 g, 2.0 mmol) in 5 mLof water is added dropwise at +1 to +5° C. to the reaction mixture toafford a precipitate of the azide 5. This azide is unstable and couldnot be isolated in pure form. IR (KBr): 3485-3290, 2928, 2866, 2270, and2134 (CON₃), 1690, 1651, 1451, 1376, 1147, 1031 cm⁻¹. TLC (EtOH-DCM 2:5)R_(f) 0.35.

8.6. Preparation of 12α-(O-Glucosyl)deoxycholic Acid-Spermine Conjugate(Comp. D)

The precipitate of azide 5 is fast filtered off through a glass filterwith porosity 40-60 μm and washed with ice water (10 mL). While stillwet, the precipitate of azide 5 is immediately transferred into asolution of spermine (0.5 g, 2.5 mmol) and triethylamine (0.5 mL) in 10mL of water. The resulting mixture is stirred for 30 min, then heated upto 60° C. for 10 min, chilled to room temperature, and treated withacetic acid to a pH 4.5-5.0. The clear solution of spermine derivative Dis purified by flash chromatography using a reverse-phase column CHP 20in MeOH-Water. The spermine derivative D is eluted with a solventgradient ranging from 50-100% of MeOH. The water-methanol fractions arecombined and concentrated. The pH is adjusted to 3.5-3.0 with HCl. Theclear solution is lyophilized to afford white, highly hygroscopic,crystalline spermine derivative D (0.37 g, yield 42% based on hydrazide4, 180° C. sinks, 200° C. decomposition). TLC (MeOH-DCM 2:8) R_(f) 0.1;(MeOH-isopropylamine-DCM 2:2:6) R_(f) 0.55. IR (KBr): 3450, 2943, 1690,1452, 1376, 1148, 1091, 950 cm⁻¹. ¹ H NMR (D₂ O): δ 4.95 (d, 1H, J=3Hz), 3.9 (s, 1H), 3.65 (m, 3H), 3.4 (m, 3H), 3.0 (m, 3H), 1.0-2.4 (m,60H), 0.95 (d, 3H), 0.90 (s, 3H), 0.62 (s, 3H). Anal. Calc. for C₄₀ H₇₄N₄ O₈. 3HCl.10H₂ O: C 46.7, H 8.91, N 5.45, Cl 10.2. Found: C 56.02, H8.91, N 5.66, C 9.47. F.W. 739.5. Found: M+Na⁺ =763.

9. Synthesis of the 7α-(O-Glucosyl)chenodeoxycholic Acid-SpermineConjugate (Comp. E of Table 1 or Comp. 6 of FIG. 4)

9.1. Preparation of 3α-(O-Anisoyl)chenodeoxycholic Acid, Methyl Ester(Comp. 1 of FIG. 4)

A mixture of methyl chenodeoxycholate (5.0 g, 12.3 mmol), anisoylchloride (2.3 g, 2.0 mL, 13.5 mmol), dimethylaminopyridine (0.8 g, 6.5mmol) in pyridine (15 mL) is heated at 100° C. for 3 h. Reaction mixtureis poured into a separatory funnel, water (200 mL) and ethyl acetate(300 mL) is added. The organic layer is washed with 5% HCl (100 mL),water (200 mL), sodium bicarbonate, and dried over sodium sulfate.Sometimes a precipitate of the product appeared between layers. Thisprecipitate may be filtered off and combined with the product that isobtained after evaporation of ethyl acetate. Total amount is 5.2 g(yield 78%, m.p. 188-190° C. from EtOH). TLC (EA-Hexane 2:5) R_(f) 0.6.IR (KBr): 3513 (OH), 2938, 2851, 1730 (COOCH₃), 1712 (Anis-CO), 1607,1579, 1509, 1451,1279, 1165, 1100, 963, 770 cm⁻¹. ¹ H NMR (CDCl ₃): δ8.03 (d, 2H), 7.96 (d, 2H), 4.85 (s, 1H), 3.85 (s, 3H), 3.65 (s, 3H),2.0-1.0 (m, 24H), 0.96 (d, 3H), 0.90 (s, 3H), 0.66 (s, 3H).

9.2. Preparation of3α-(O-Anisoyl)-7α-(tetra-O-benzyl-O-glucosyl)chenodeoxycholic Acid,Methyl Ester (Comp. 2 of FIG. 4)

Triflic anhydride (2.1 g, 1.27 mL, 7.4 mmol) is added to dry toluene(100 mL), chilled up to -72 to -75° C. with acetone-dry ice bath.Phenylsulphenyl glucoside (5.1 g, 7.4 mmol) in 20 mL of dry toluene isadded dropwise, then in 10 mins the 2,6-di-tert-butyl-4-methyl-pyridine(1.52 g, 7.4 mmol) in toluene (15 mL) is added, and in 5 min the anisoylderivate 1 (3.2 g, 5.9 mmol in 30 mL of dry toluene) is added dropwise.When TLC showed the starting material disappeared, saturated solution ofthe sodium bicarbonate (150 mL) is poured, and the mixture istransferred into a separatory funnel. The organic layer is washed withwater (20 mL), brine (50 mL), dried over sodium sulfate, andconcentrated to give a thick oil. It is purified by flash chromatography(EA-Hexane); the product is eluted with 20% ethyl acetate. The product(4.0 g, yield 62%) is obtained as a thick colorless oil. TLC (EA-Hexane2:5) R_(f) 0.65. IR (neat): 2950, 2870, 1690, 1745, 1610, 1450,1275,1160, 1050, 970, 775 cm⁻¹.

9.3. Preparation of 3α-(Anisoyl)-7α-(O-glucosyl)chenodeoxycholic Acid,Methyl Ester (Comp. 3 of FIG. 4)

The above obtained oil (4.0 g, 3.7 mmol) is dissolved in ethyl acetate(15 mL) and ethanol (75 mL), together with catalyst (Pd(OH)₂ /C, 2.0 g).Formic acid (2.0 mL) is added to the mixture. The mixture is set up forhydrogenation in an 0.5 L Parr's apparatus at 50 psi for 24 h. Thecatalyst is filtered off, and the filtrate is evaporated to give acrystalline residue of 3 (1.8 g, yield 69%), m.p. 258-260° C. (fromEtOH), no decomposition. TLC (MeOH-DCM 1:9) R_(f) 0.35. IR (KBr): 3439(OH), 2863, 1742 (COOCH₃), 1684 (anis. CO), 1606, 1284,1260, 1022, 967,773 cm⁻¹. ¹ H NMR (CDCl₃): δ 7.9 (d, 2H, J=6 Hz), 6.8 (d, 2H, J=6 Hz),4.95 (d, 1H, J=3 Hz), 4.75 (s, 1H), 3.80 (s, 3H), 3.58 (s, 3H), 3.3-3.5(m, 4H), 2.0-1.1 (m, 30H), 0.92 (s, 3H), 0.88 (d, 3H), 0.62 (s, 3H).

9.4. Preparation of 7α-(O-Glucosyl) chenodeoxycholic Acid, Hydrazide(Comp. 4 of FIG. 4)

The methyl ester 3 (1.7 g, 3.0 mmol) is refluxed in mixtureEtOH-hydrazide hydrate (20 mL+6 mL) for 2 h. The crystals of hydrazide 4(0.45 g, m.p. 238-40° C.) that form are separated from solution at roomtemperature and filtered off. The mother liquid is concentrated, toafford an additional amount of hydrazide 4 (0.65 g). Total yield 1.1 g(70%). TLC (MeOH-DCM, 2:8) R_(f) 0.05. IR(KBr): 3378 (NH, OH), 2927,1697 (CONH), 1601,1260, 1020, 980, 770 cm⁻¹.

9.5. Preparation of 7α-(O-Glucosyl)Chenodeoxycholic Acid, Azide (Comp. 5of FIG. 4)

Hydrazide 4 (0.8 g, 1.4 mmol) is dissolved in 10 mL 10% HCl, chilled to+3 to +5° C., then NaNO₂ (0.21 g, 3 mmol) in 5.0 mL of water is addeddropwise affording a precipitate of azide 5. This compound is unstableand cannot be isolated as a pure substance. TLC (EtOH-DCM 2:8) R_(f)0.45. IR (KBr): 3490-3300, 2930, 2850, 2260 and 2133 (CON₃), 1700, 1640,1450, 1366, 1147, 1050 cm⁻¹.

9.6. Preparation of 7α-(O-Glucosyl)chenodeoxycholic Acid-SpermineConjugate (Comp. E)

The precipitate of azide 5 is fast filtered through a glass filter(porosity 40-60 μm) washed with ice water (5 mL), and while wet isimmediately transferred into a solution of spermine (0.5 g, 2.5 mmol)and triethylamine (0.5 mL) in 10 mL of water. The mixture is stirred for30 min, then is heated up to 60° C. for 10 min, then is chilled to roomtemperature. The pH is adjusted to 4.5-5.0 using acetic acid. Theinsoluble impurities are filtered off, and the clear filtrate ofspermide E is purified by flash chromatography using a reverse-phasecolumn CHP-20. The spermide E is eluted with a solvent gradient rangingfrom 40-100% of MeOH. The water-methanol fractions are combined,evaporated to dryness. Water (10 mL) and concentrated HCl (0.2 mL) isadded, and the clear solution is lyophilized to afford white, highlyhygroscopic, crystalline spermide E (0.50 g, yield 42% based onhydrazide 4, m.p. 162-164° C. with decomp.). TLC (MeOH-i-PrOH-DCM 2:2:6)R_(f) 0.6. IR (KBr): 3447, 2934, 2865, 1652 (CONH), 1457, 1379,1256,1026, 772 cm⁻¹. ¹ H NMR (D₂ O): δ 4.85 (d, 1H, J=3 Hz), 3.5-3.8 (m, 8H),3.5 (m, 6H), 3.1 (m, 2H), 2.9-3.0 (m, 10H), 2.1-1.0 (m, 40H), 0.796 (m,6H), 0.551 (s, 3H). Anal. Calc. for C₄₀ H₇₄ N₄ O₈.3HCl.10H₂ O: C 46.7,H9.44, N 5.45, Cl 10.37. Found: C 60.8, H 8.97, N 4.60, Cl 6.09. F.W.847.5. Mass-spectrum Fab.M-HCl+H⁺ =815. Found: 815.

10. Synthesis of 3α,7α,12α-Trihydroxy-5β-cholan-24oic Acid,N-Oxysuccinimide (Comp. 1 of FIG. 5)

A mixture of dry cholic acid (8.16 g, 20 mmol), dicyclohexeylcarbodimide(4.33 g, 21 mmol) and N-hydroxysuccinimide (2.417 g, 21 mmol) is stirredin dry methylene chloride (200 mL) at room temperature for 6 h. Thereaction mixture is filtered, and the filtrate concentrated. The residueis purified by flash chromatography through florosil (EtOH:CH₂ Cl₂ 1:19)giving 8 g (79% yield) of compound 1 as a white foam (mp 92-95° C). TLC(EtOH:CH₂ Cl₂ 1:19) R_(f) 0.6. IR (KBr): 3385 (br), 2933, 2861, 2118,1814, 1783, 1738, 1376, 1208, 1073 cm⁻¹. ¹ H NMR (CDCl₃): δ 3.94 (s,1H), 3.81 (s, 1H), 3.42 (m, 1H), 2.82 (br, 4H), 2.30-1.00 (m, 24H), 0.99(d, 1H, J=5.7 Hz), 0.862 (s, 3H), 0.67 (s, 3H). Fab MS: 528 (M+Na)⁺.

10.1. Preparation of 3α,7α,12α-Trihydroxy-5β-cholan-24-oic Acid,N-(4,9-Diaza-12-aminododecyl)amide (Comp. B of Table 1 or Comp. 2 ofFIG. 5)

To a stirred solution of spermine (303 mg, 1.5 mmol) and triethylamine(1 mL) in anhydrous methylene chloride (20 mL), N-oxysuccinimidocholate1 (505 mg, 1 mmol) in anhydrous methylene chloride (20 mL) is addeddropwise during a 10 min period. The solution is then stirred for 3 h atroom temperature. The reaction mixture is filtered and filtrateconcentrated. The residue is purified by flash chromatography usingCHP-20 reverse-phase resin (water and then 75% aqueous MeOH), affording2 (360 mg, 52% yield) as a white foam (mp 140-145° C.). TLC (MeOH:CH₂Cl₂ :isopropylamine 4.5:4.5:1) R_(f) 0.4. IR (KBr): 3350 (br), 2934,2859, 1685, 1644, 1547, 1449, 1377,1234,1207, 1078, 1046 cm⁻¹. ¹ H NMR(DMSO-d₆ and 2 drops of D₂ O): δ 3.78 (s, 1H), 3.61 (s, 1H), 3.40-2.80(m, 9H), 2.42-0.77 (m, 42H), 0.55 (s, 3H). Fab MS: 615 (M+Na)⁺.

11. Synthesis of 3α,12α-Dihydroxy-7-deoxy-5β-cholan-24-oic Acid,N-Oxysuccinimide

A mixture of dry deoxycholic acid (2.356 g, 6 mmol),dicyclohexeylcarbodimide (1.444 g, 7 mmol) and N-hydroxy-succinimide(0.806 g, 7 mmol) are stirred in dry methylene chloride (200 mL) at roomtemperature for 6 h. The reaction mixture is filtered, and the filtrateconcentrated. The residue is purified by flash chromatography throughflorosil (EtOH:CH₂ Cl₂ 1:19), affording 1.764 g (60% yield) of the titlecompound as a white foam (mp 75-80° C.). TLC (EtOH:CH₂ Cl₂ 1:9) R_(f)0.5. IR (KBr): 3364 (br), 2934, 2862, 1814, 1783, 1738, 1655, 1627,1449, 1376, 1208, 1068 cm⁻¹. ¹ H NMR (CDCl₃): δ 3.97 (s, 1H), 3.62 (m,1H), 2.82 (br, 4H), 2.70-0.83 (m, 30H), 0.67 (s, 3H). Fab MS: 512(M+Na)⁺.

11.1. Preparation of 3α,12α-Dihydroxy-7-deoxy-5β-cholan-24-oic Acid,N-(12-Aminododecane)amide

To a stirred solution of dodecan-1,12-diamine (600 mg, 3 mmol) andtriethylamine (1 mL) in anhydrous methylene chloride (25 mL),N-oxysuccinimidodeoxycholate (980 mg, 2 mmol) in anhydrous methylenechloride (25 mL) is added dropwise during 10 minute period. The contentsare stirred for 14 h at room temperature. The reaction mixture isfiltered, and the filtrate concentrated. The residue is purified byflash chromatography using CHP-20 reverse-phase resin (20%, 40%, 60%,80% aqueous MeOH and then MeOH) to give the title compound (575 mg, 50%yield) as a white foam (mp 118-120° C.). TLC (MeOH:CH₂ Cl₂:isopropylamine 4.5:4.5:1) Rf 0.8. IR (KBr): 3365 (br), 2928, 2857,1654, 1647, 1534, 1449, 1376, 1044 cm⁻¹. ¹ H NMR (CDCl₃): δ 3.97 (s,1H), 3.62 (m, 1H), 3.21 (q, 1H, J=6.6 Hz), 2.70-1.00 (m, 48H), 0.98 (d,1H, J=6.0 Hz), 0.90 (d, 1H), 0.67 (s, 3H). Fab MS: 622 (M+2Na)⁺.

12. Preparation of Bis(glycosylated)cholic Acid-Spermine Conjugate(Comp. C of Table 1 or Comp. 7 of FIG. 6)

12.1. Synthesis of 3α-Hydroxy-7α,12α-di(2',3',4',6'-tetra-O-benzyl-1'α-glucosyl)-5β-cholan-24-oic Acid,N-Oxysuccinimide (Comp. 5 of FIG. 6)

A solution of dry7α,12α-di-(2',3',4',6'-tetra-O-benzyl-1'α-glucosyl)-5.beta.-cholan-24oic acid (1.452 g, 1 mmol), N-hydroxysuccinimide (126 mg, 1.1 mmol) andDCC (226 mg, 1.1 mmol) in dry methylene chloride is stirred at roomtemperature for 3 h. The reaction mixture is filtered, and the filtrateconcentrated. The residue is purified by flash chromatography through acolumn of florosil (EtOH:CH₂ Cl₂ 1:19) to give 1.40 g (90% yield) ofcomp. 5 as a white foam (mp 63-65° C.). TLC (EtOH:CH₂ Cl₂ 1:19) R_(f)0.5. IR (KBr): 3062, 3030, 2928, 2863, 2117, 1813, 1784, 1740, 1685,1496, 1453, 1363,1206, 1070 cm⁻¹. ¹ H NMR (CDCl₃): δ 7.40-6.90 (m, 40H),5.10-3.10 (m, 33H), 2.80 (br s, 4H), 2.62-0.84 (m, 30H), 0.73 (s, 3H).Fab MS: 1572 (M+Na)⁺.

12.2. Preparation of 3α-Hydroxy-7α,12α-di(2',3',4',6'-tetra-O-benzyl-1'α-glucosyl)-5β-cholan-24-oic Acid,N-(4,9-Diaza-12-aminododecyl)amide (Comp. 6 of FIG. 6)

To a stirred solution spermine (0.808 g, 4 mmol) and triethylamine (3mL) in dry methylene chloride (50 mL), comp. 5 (5.16 g, 3.33 mmol) inmethylene chloride (50 mL) is added and stirred for 4 h. The reactionmixture is filtered, and the filtrate is washed with water (2×50 mL),dried (Na₂ SO₄), and concentrated. The residue is purified by flashchromatography through a column of CHP-20 reverse-phase resin (water,then methanol) to afford comp. 6 (4.9 g, 85% yield) as a white foam (mp58-60° C.). TLC (MeOH:CH₂ Cl₂ :isopropylamine 4.5:4.5:1) R_(f) 0.2. IR(KBr): 3063, 3030, 2928, 2863, 1655, 1628, 1496, 1452, 1362, 1208, 1147,1070, 1028 cm⁻¹. ¹ H NMR (CDCl₃): δ 7.40-6.90 (m, 40H), 6.62 (br s, 1H),5.03-3.20 (m, 33H), 3.00-0.86 (m, 55H), 0.72 (S, 3H). Fab MS: 1659(M+Na)⁺. Anal. Calc. for C₁₀₂ H₁₃₂ O₁₄ N₄.H₂ O: C, 74.16; H, 8.19; N,3.35. Found: C, 73.53; H, 8.24; N, 3.72.

12.3. Preparation of 3α-Hydroxy-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oicAcid, N-(4, 9-Diaza-12-aminododecyl)amide (Comp. C)

To a solution of comp. 6 (2.455 g, 1.5 mmol) and 1N aqueous HCl (25 mL)in THF (50 mL), 20% palladium hydroxide on carbon (2 g, Perlman'scatalyst) is added. The mixture is subjected to hydrogenalysis at 50 psifor 6 h. The reaction mixture is filtered through sand and membranefilter and concentrated. The residue is dissolved in water (5 mL) andfiltered. The filtrate is purified by flash chromatography through acolumn of CHP-20 reverse-phase column (water, followed by MeOH:Water1:9) to give 1.078 g (70% yield) of C as a white foam (mp 83-85° C.).TLC (trifluoroacetic acid:water 1:9) R_(f) 0.35. IR (KBr): 3365 (br),2938, 2867, 1638, 1629, 1561, 1545, 1459, 1150, 1075, 1048, 1025 cm⁻¹. ¹H NMR (D₂ O): δ 5.06 (d, 1H, J=3.6 Hz), 4.85 (d, 1H, J=3.6 Hz), 3.95 (brs, 1H), 3.78-2.88 (m, 21H), 2.28-0.76 (m, 46H), 0.64 (s, 3H). Fab MS:940 (M+Na)⁺. Anal. Calc. for C₃₆ H₈₄ O₁₄ N₄.3HCl.5H₂ O: C, 49.66; H,8.52; N, 5.04; Cl, 9.44. Found: C, 49.68; H, 8.60; N, 5.06; Cl, 9.65.

13. Synthesis of Various (Polyaminoalkyl)amides of Deoxycholic andChenodeoxycholic Acids

13.1. Preparation of 3α,12α-Dihydroxy-7-deoxy-5β-cholan-24-oic Acid,N-(3,6,9-Triaza-11-aminoundecyl)amide (Comp. F of Table 1)

To a solution of tetraethylenepentamine (0.378 g, 2.5 mmol) andtriethylamine (0.3 mL) in DMF (5 mL) is added dropwise over 10 min theN-oxysuccinimidodeoxycholate (1.0 g, 2 mmol) in 5 mL of DMF. Thesolution is stirred overnight at room temperature, poured into water (20mL). The precipitate obtained is washed with cold water (50 mL),dissolved in 10 mL of 2% HCl, and filtered. The solution is poured overa CHP-20 reverse phase column and eluted using a 40-80% MeOH in watersolvent gradient system to afford 1.1 g (72% yield) of thetrihydrochloride, pentahydrate form of the title compound, as a whitepowder after lyophilization (m.p. 130-132° C.). TLC (MeOH:i-PrNH₂ :DCM2:2:6) R_(f) 0.6. IR (KBr): 3419, 2934, 1642 (CONH--), 1553, 1454, 1038cm⁻¹. ¹ H NMR (D₂ O): δ 3.88(s, 1H), 2.9-3.3 (m, 16H), 1.2-2.4 (m, 42H),0.88 (d, 3H), 0.78 (s, 3H), 0.55 (s, 3H). Fab MS:696 (Base.3HCl+Na⁺).Anal. Calc. for C₃₂ H₆₁ N₅ O₃.3HCl.5H₂ O: C 50.3; H 9.69; N 9.17; Cl13.95. Found: C 51.5; H 9.04; N 10.1; Cl 10.9.

13.2. Preparation of 3α,12α-Dihydroxy-7-deoxy-5β-cholan-24-oic Acid,N-(3, 6, 9 ,12-Tetraaza-14-aminotetradecyl)amide (Comp. G of Table 1)

To a solution of pentaethylenehexamine (0.58 g, 2.5 mmol) andtriethylamine (0.3 mL) in DMF (5 mL) is added dropwise over 10 min theN-oxysuccinimidedeoxycholate (1.0 g, 2 mmol) in 5 mL of DMF. Thesolution is stirred overnight at room temperature, then poured intowater (50 mL) to give a precipitate. The liquid phase is decanted. Thesemi-solid precipitate is washed successively with cold 5% NaOH (10mL×2) and water (10 mL), dissolved in 10 mL of 10% acetic acid, andpurified by flash chromatography through a CHP-20 reverse-phase columnusing a 40-100% MeOH in water solvent gradient system. The fractionscontaining product are combined, evaporated at reduced pressure,dissolved in 2% aqueous HCl solution, and lyophilized to afford 0.75 g(42% yield) of the title compound as a white powder (m.p. 140-142° C.).TLC (MeOH:i-PrNH₂ :DCM 2:2:6) R_(f) 0.65. IR (KBr): 3425, 2932, 1770(COOH), 1643 (CONH), 1552 (COO⁻), 1454, 1032 cm⁻¹. ¹ H NMR (D₂ O) δ 3.92(s, 1H), 2.6-3.6 (m, 20H), 1.0-1.6 (m, 30H), 0.83 (d, 3H), 0.75 (s, 3H),0.55 (s, 3H). Fab MS: 863 (M+H⁺). Anal. Calc. for C₃₄ H₆₆ N₆O₃.2HCl.3AcOH: C 55.8; H 9.28; N 9.70; Cl 8.2. Found: C 59.0; H 9.40; N8.3; Cl 6.6.

13.3. Preparation of 3α,7α-Dihydroxy-12-deoxy-5β-cholan-24-oic Acid,N-(4,9-Diaza-12-aminododecyl)amide (Comp. H of Table 1)

To a solution of spermine (0.8 g, 2 mmol) and triethylamine (0.3 mL) in5 mL of DMF is added dropwise the N-oxysuccinimidechenodeoxycholate (1.0g, 2 mmol) in 5 mL of DMF. The mixture is stirred overnight at roomtemperature, then poured into DCM (100 mL). The precipitate of thehydroxysuccinimide is filtered, and the filtrate is evaporated to give aliquid phase, which is poured into water (100 mL). The precipitate ofthe product is obtained. It is dissolved in MeOH (5 mL) and passedthrough a CHP-20 reverse-phase column. A 30% MeOH in water solventsystem is used to elute the product. The solvent is removed byevaporation, and the residue is dissolved in 1 mL of trifluoroaceticacid. The resulting solution is diluted up to 10 mL with water,filtered, and the filtrate subsequently lyophilized to afford 0.9 g (50%yield) of a solid (m.p. 96-100° C.). The product is soluble in water. A5% solution of the trifluoroacetate salt of the chenodeoxycholicacid-spermine conjugate is stable at room temperature over about 12-24h, after which a precipitate of the base separates as a slurry. TLC(MeOH:i-PrNH₂ :DCM 1:1:2) R_(f) 0.7. IR (KBr): 3406, 2939, 2869, 1778(COOH), 1680 (CONH--), 1553, 1458, 1196, 834, 722 cm⁻¹. ¹ H NMR (D₂ O):δ 3.75 (s, 1H), 3.4 (s, 1H), 2.8-3.15 (m, 12H), 2.2-1.2 (m, 39H), 0.9(d, 3H), 0.86 (s, 3H), 0.55 (s, 3H). Fab MS: (M+Na⁺)=598. Anal. Calc.for C₃₄ H₆₄ N₄ O₃.3CF₃ COOH: C 52.5; H 7.29; N 6.09. Found: C 53.5; H7.20; N 4.95.

13.4. Preparation of 3α,7α-Dihydroxy-12-deoxy-5β-cholan-24-oic Acid,N-(3,6,9-Triaza-12-aminoundecyl)amide (Comp. I of Table 1)

To a solution of the tetraethylenepentaamine base (1.90 g, 10 mmol) andtriethylamine (1.0 g, 10 mmol) in 100 mL of DCM,N-oxysuccinimidechenodeoxycholate (2.46 g, 5 mmol) in DCM (50 mL) isadded and the solution is stirred 48 h at room temperature. The reactionmixture is diluted with 100 mL of DCM, washed with water (2×100 mL),dried over sodium sulfate and evaporated to dryness. The residue isdissolved in 25 mL of 10% acetic acid, filtered and the clear filtrateis purified on CHP-20 column in MeOH-water. At 40%-80% of MeOH theproduct is eluted. The combined fractions are acidified by 10% HCl (5mL) and the methanol is distilled off under vacuum. The rest of thewater solution is removed by lyophilization to give 2.84 g (77% yield,m.p. 200-203° C. decomp.) of the pentaaminotetraethyleneamide of thechenodeoxycholic acid. TLC (MeOH:i-PrNH₂ :DCM) R_(f) 9.8. IR (KBr):3350, 2974, 1665, 1635, 1551, 1539, 1460, 1470, 1377, 1077, 978, 766cm⁻¹. ¹ H NMR (D₂ O)=δ 3.378 (s, 1H), 2.9-3.4 (m, 16H), 1.8-1.2 (m,39H), 0.85 (d, 3H), 0.76 (s, 3H), 0.55 (s, 3H). Fab MS: (M+H⁺)=564.Anal. Calc. for C₃₂ H₆₁ N₅ O₃.4HCl.2H₂ O: C 51.54; H 9.26; N 9.39; Cl19.06. Found: C 50.48; H 8.84; N 8.86; Cl 19.7.

13.5. Preparation of 3α,7α,12α-Trihydroxy-5β-cholan-24-oic Acid,N-(3,6,9,-Triaza-12-aminoundecyl)amide (Comp. J of Table 1)

To a solution of the tetraethylenepentamine (0.8 g, 5 mmol) and TEA (0.3g, 3 mmol) in DCM (25 mL) the N-oxysuccinimidecholate (1.0 g, 2.0 mmol)is added. A clear solution is stirred at room temperature for 48 h, thereaction mixture is diluted with DCM (100 mL), washed with cold water(20 mL), dried over sodium sulfate and evaporated to dryness. Theresidue is dissolved in 20 mL of 5% AcOH. A purification is carried outon a CHP-20 reverse phase column in MeOH-water. The product runs at40%-80% of MeOH. The fractions containing target compound are combined,methanol is distilled off, and 10 mL of 10% HCl is added. Lyophilizationgives 0.70 g (50% yield) of the pure substance, m.p. 135-140° C. TLC(MeOH:i-PrNH₂ :DCM-1:1:3) R_(f) 0.8. IR (KBr): 3406, 2937, 1640 (C═O),1556, 1453, 1376, 1023 cm⁻¹. ¹ H NMR (D₂ O): δ 3.8 (s, 1H), 3.65 (s,1H), 3.0-3.3 (m, 16H), 2.0-1.1 (m, 26H), 0.78 (d, 3H), 0.72 (s, 3H),0.48 (s, 3H). Fab MS: (M+H⁺)580. Anal. Calc. for C₃₂ H₆₁ N₅ O₄.5HCl: C50.4; H 8.66; N 9.18; Cl 23.29. Found: C 47.27; H 8.31; N 8.57; Cl25.63.

13.6. Preparation of 3α,7α,12α-Trihydroxy-12-deoxy-5β-cholan-24-oicAcid, N-(3, 6, 9,12-Tetraaza-14-aminotetradecyl)amide (Comp. K of Table1)

To a solution of the pentaethylenehexamine (0.9 g, 5.5 mmol) andtriethylamine (0.3 g, 3 mmol) in DCM (10 mL) the neatN-oxysuccinimidecholate (1.0 g, 2.0 mmol) is added with stirring at roomtemperature. The reaction mixture is stirred at room temperature for 48h. At the end of this period, the reaction mixture turns into asemisolid, which is diluted with 150 mL of DCM, washed with cold water(2×50 mL), dried and distilled to dryness, dissolved in 10 mL of 10%AcOH, filtered from insoluble material, and purified on a reverse-phasecolumn CHP-20 with methanol-water. The product runs at 40%-70% ofmethanol. The combined fractions containing product is distilled frommethanol. Afterwards, 10% HCl (5 mL) is added. After lyophilization,0.96 g (55% yield) is obtained (m.p. 230° C., decomp.). TLC(DCM:MeOH:iPrNH₂ -5:1:1) R_(f) 0.85. IR (KBr): 3393, 2937, 1646 (C═O),1550, 1483, 1376, 1072, 1028, 774 cm⁻¹. ¹ H NMR (D₂ O): δ 3.83 (s. 1H),3.67 (s. 1H), 3.1-3.5 (m. 21H), 2.0-1.4 (m. 26H), 0.78 (d. 3H), 0.68 (s.3H), 0.48 (s. 3H). Fab MS: (M+H⁺): 623. Anal. Calc. for C₃₂ H₆₁ N₅O₄.5HCl: C 50.4; H 8.66; N 9.18; Cl 23.29. Found: C 47.27; H 8.31; N8.57; Cl 25.63.

13.7. Preparation of 3α-Hydroxy-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oicAcid, N-(3,6,9,12-Tetraaza-14-aminotetradecyl)amide (Comp. L of Table 1)

13.7.1 Preparation of 3α-Hydroxy-7α,12α-di(2', 3', 4',6'-tetra-O-benzyl-1α-glucosyl)-5β-cholan-24-oic Acid,N-(3,6,9,12-Tetraaza-14-amino-tetradecyl)amide

To a stirred solution of pentaethylenehexamine (367 mg, 1.5 mmol) andtriethylamine (2 mL) in dry methylenechloride (50 mL),N-oxysuccinimide-7α,12α-di(perbenzylglucosyl)cholate (1.549 g, 1 mmol)in methylene chloride (50 mL) is added dropwise and stirred for 48 h.The reaction mixture is filtered, and the filtrate is concentrated. Theresidue is purified on flash chromatography over CHP-20 reverse phaseresin (eluants, water and then gradually increasing to 90% methanol;product is obtained from 90% methanol in water fractions) affords thetitle compound (950 mg, 57% yield) as a white foam (m.p. 78-80° C.). TLC(solvent-MeOH:CH₂ Cl₂ :Isopropylamine 4:4:2) R_(f) 0.1. IR (KBr): 3500(br), 3086, 3061, 3030, 2929, 2864, 1699, 1652, 1453, 1363, 1155, 1071,1028 cm⁻¹. ¹ H NMR (CDCl₃): δ 7.40-6.90 (m, 40H), 5.03-3.10 (m, 33H),2.90-0.66 (m, 65H). Fab MS: 1674 (M+Na)⁺.

13.7.2 Preparation of 3α-Hydroxy-7α,12α-di(1α-glucosyl)-5β-cholan-24-oicAcid, N-(3, 6, 9, 12-Tetraaza-14-aminotetra-decyl)amide

To a solution of the compound from above (333 mg, 0.2 mmol) and 1Naqueous HCl (3 mL, 3 mmol) in THF and water (2:1, 30 mL), 20% palladiumhydroxide on carbon (300 mg, Perlman's catalyst) is added and themixture is subjected to hydrogenolysis at 50 psi for 15 h. The reactionmixture is filtered through sand and membrane filter and thenconcentrated. The residue is dissolved in water (5 ml) and filtered. Thefiltrate is purified on flash chromatography over CHP-20 reverse phasecolumn (water, followed by MeOH:Water=1:19, 1:4 and 2:3; product isfound in 20% methanol in water fractions) to give 110 mg (40% yield) ofthe desired compound as a white foam (mp 180-82° C.). TLC(solvent-Trifluoroacetic acid:Water 1:9) R_(f) 0.3. IR (KBr): 3394,2934, 2867, 1652, 1647, 1636, 1558, 1541, 1027 cm⁻¹. ¹ H NMR (D₂ O): δ5.09 (d, 1H, J=3.6 Hz), 4.86 (d, 1H, JjJJ-3.6 Hz), 3.95 (brs, 1H),3.80-255 (m, 15H), 2.30-0.65 (m, 56H). Fab MS: 970 (M+Na)⁺. Anal. Calc.for C₄₆ H₈₆ O₁₄ N₆.4HCl: C 50.55; H 8.30; N 7.69; Cl 12.97. Found: C50.67; H 8.71; N 6.70; Cl 11.65.

13.8. Preparation of 3α-Hydroxy-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oicAcid, N-(3,6,9-Triaza-12-aminoundecyl)amide (Comp. M of Table 1)

13.8.1. Preparation of3α-Hydroxy-7α,12α-di-(2',3',4',6'-tetra-O-benzyl-1'.alpha.-glucosyl)-5β-cholan-24-oicAcid, N-(3,6,9-Triaza-12-aminoundecyl)amide

To a stirred solution of tetraethylenepentamine (285 mg, 1.5 mmol) andtriethylamine (2 mL) in dry methylene chloride (50 mL),N-oxysuccinimide-7α,12α-di (perbenzylglucosyl) cholate (1.549 g, 1 mmol)in methylene chloride (50 mL) is added dropwise and stirred for 48 h.The reaction mixture is filtered, and the filtrate is concentrated. Theresidue is purified on flash chromatography over CHP-20 reverse phaseresin (eluants, water and then gradually increasing to 90% methanol;product is obtained from 90% methanol in water fractions) to afford thetitle compound (1 g, 63.8% yield) as a white foam (mp 74-76° C.). TLC(solvent-MeOH:CH₂ Cl₂ :isopropylamine 4:4:2) R_(f) 0.1. IR (KBr): 3365,3086, 3061, 3029, 2925, 2864, 1699, 1653, 1496, 1453, 1155, 1070, 1028cm⁻¹. ¹ H NMR (CDCl₃): δ 7.40-6.95 (m, 40H), 5.10-3.20 (m, 33H),2.82-0.82 (m, 57H), 0.72 (s, 3H). Fab MS: 1651 (M+Na)⁺.

13.8.2. Preparation of3α-Hydroxy-7α,12α-di-(1'α-glucosyl)-5β-cholan-24-oic Acid, N-[3, 6,9,-Triaza-12-aminoundecyl)amide

To a solution of the above compound (486 mg, 0.3 mmol) and 1N aqueousHCl (4 mL, 3 mmol) in THF and water (2:1, 30 mL), 20% palladiumhydroxide on carbon (400 mg, Perlman's catalyst) is added and themixture is subjected to hydrogenolysis at 50 psi for 15 h. The reactionmixture is filtered through sand and membrane filter and concentrated.The residue is dissolved in water (5 mL) and filtered. The filtrate ispurified on flash chromatography over CHP-20 reverse phase column (waterfollowed by MeOH:water=1:19, 1:4 and 2:3; product is found in 20%methanol in water fractions) to give 160 mg (50% yield) of the desiredcompound as white foam (m.p. 151-53° C.). TLC (solvent-Trifluoroaceticacid:water 1:9) R_(f) 0.3. IR (KBr): 3390, 2938, 2869, 1652, 1647, 1636,1541, 1457,1251, 1150, 1073, 1026 cm⁻¹. ¹ H NMR (D₂ O):δ 5.09 (br s,1H), 4.86 (br s, 1H), 4.00 (m, 2H), 3.85-2.60 (m, 16H), 2.30-0.75 (m,49H) and 0.66 (s, 3H). Fab MS: 927 (M+Na)⁺. Anal. Calc. for C₄₄ H₈₁ O₁₄N₅.3HCl: C 52.14; H 8.35; N 6.91; Cl 10.49. Found: C 52.41; H 8.75; N5.21; Cl 9.49.

13.9. Preparation of 3α-Hydroxy-7,12-dideoxy-5β-cholan-24-oic acid,N-(3,6, 9,12-Tetraaza-14-aminotetrodecyl)amide (Comp. N of Table 1)

N-oxysuccinimidelithocholate (1.0 g, 2.1 mmol) is added topentaethylenehexamine (0.73 g, 3.2 mmol) and triethylamine (0.21 g, 2.1mmol) in DCM (50 mL). The reaction mixture is stirred at roomtemperature for 48 h, diluted with DCM (100 mL), washed with water(2×100 mL), dried, and the solvent evaporated. The residue is dissolvedin 50 mL of 10% AcOH over 5 h with vigorous stirring. The cloudysolution is purified on a reverse phase column in MeOH-water. Afterlyophilization, 1.1 g (60% yield) of the product is obtained (m.p. 94°C.). TLC (DCM:MeOH:iPrnH₂ 5:1:1) R_(f) 0.65. IR (KBr): 3390, 2933, 2862,1648 (C═O), 1555, 1402, 1075, 656 cm⁻¹. ¹ H NMR (D₂ O): δ 3.2-2.6 (m,19H), 1.7-1.0 (m, 29H), 0.70(s, 6H), 0.42 (s, 3H). Fab MS: (597). Anal.Calc. for C₃₄ H₆₆ N₆ O₂.5AcOH: C 59.3; H 9.66; N 9.44. Found. C 58.2; H9.51; N 10.9.

14. Synthesis of 3β-Amino-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oic Acid,N-(4,9-Diaza-12-amino-dodecyl)amide-HCl Salt

14.1. Preparation of3β-Azido-7α,12α-di(2',3',4',6'-tetra-O-benzyl-1α-glucosyl)-5β-cholan-24-oicAcid, N-Oxysuccinimide

A solution of dry 3-β-azido-7α,12α-di-(2',3',4',6'-tetra-O-benzyl-1α-glucosyl)-5β-cholan-24-oic acid(4.443 g, 3 mmol), N-hydroxysuccinimide (406 mg, 3.5 mmol) and DCC (722mg, 3.5 mmol) in dry methylene chloride is stirred at room temperaturefor 3 h. The reaction mixture is filtered, and the filtrateconcentrated. The residue is purified by flash chromatography through aflorosil column (EtOAc:Hexane 1:3) to give 4 g (80% yield) of theactivated cholate ester as a white foam (m.p. 64-66° C.). TLC(EtOAc:Hexane 3:7) R_(f) 0.3. IR (KBr): 3325, 3088, 3062, 3030, 2924,2867, 2099, 1815, 1785, 1742,1206, 1070 cm⁻¹. ¹ H NMR (CDCl₃): δ7.40-6.90 (m, 40H), 5.02 (q, 2H, J=3.6 Hz), 4.90-3.42 (m, 31H), 2.80 (brs, 4H), 2.62-0.90 (m, 30H), 0.75 (s, 3H).

14.2. Preparation of3β-Azido-7α,12α-di(2',3',4',6'-tetra-O-benzyl-1'α-glucosyl)-5β-cholan-24-oicAcid, N-(4,9-Diaza-12-aminododecyl)amide

To a stirred solution of spermine (0.303 g, 1.5 mmol) and triethylamine(3 mL) in dry methylene chloride (75 mL), compound from 14.1 (1.579 g, 1mmol) in methylene chloride (75 mL) is added and stirred for 4 h. Thereaction mixture is filtered, and the filtrate is washed with water(2×50 mL), dried (Na₂ SO₄), and concentrated. The residue is purified byflash chromatography through a CHP-20 reverse-phase resin (eluant: waterand then methanol) to afford the title compound (1.46 g, 86% yield) as awhite foam (m.p. 60-62° C.). TLC (MeOH:CH₂ Cl₂ :isopropylamine4.5:4.5:1) R_(f) 0.5. IR (KBr): 3432 (br), 3087, 3062, 3030, 2925, 2865,2098, 1670, 1663, 1656, 1640, 1630, 1496, 1452, 1364, 1071, 1028 cm⁻¹. ¹H NMR (CDCl₃): δ 7.40-6.90 (m, 40H), 6.30-6.10 (m, 1H), 5.04-3.10 (m,33H), 2.80-0.83 (m, 55H), 0.73 (s, 3H).

14.3. Preparation of3β-Amino-7α,12α-di(2',3',4',6'-tetra-O-benzyl-1'α-glucosyl)-5β-cholan-24-oicAcid, N-(4,9-Diaza-12-aminododecyl)amide

To a stirred mixture of the compound of 14.2 (0.999 g, 0.6 mmol) andRaney Ni (500 mg) in ethanol (10 mL) is added dropwise over 10 min ahydrazine hydrate (0.2 mL, 4 mmol) in ethanol (10 mL). The mixture isstirred for 2 h, after which it is filtered. The filtrate isconcentrated under vacuum (aspirator pump). The residue is washed withwater (3×50 mL) and dried under vacuum to give the desired 3-aminocompound (920 mg, 94%) as a white foam (m.p. 55-57° C.). TLC (MeOH:CH₂Cl₂ :isopropylamine 4.5:4.5:1) R_(f) 0.5. IR (KBr): 3415 (br), 3087,3062, 3029, 2925, 2864, 1669, 1662, 1654, 1647, 1630, 1496, 1453, 1362,1086, 1070, 1028 cm⁻¹. ¹ H NMR (CDCl₃): δ 7.40-6.90 (m, 40H), 6.30-6.10(m, 1H), 5.00-3.00 (m, 33H), 2.80-0.78 (m, 55H), 0.66 (s, 3H).

14.4. Preparation of 3β-Amino-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oicAcid, N-(4 , 9-Diaza-12-aminododecyl)amide.HCl Salt

To a solution of compound of 14.3 (0.91 g, 0.56 mmol) and 1N aqueous HCl(8 mL, 8 mmol) in THF (25 mL) and water (10 mL) is added 20% palladiumhydroxide on carbon (0.9 g, Perlman's catalyst), and the mixture issubjected to hydrogenolysis at 50 psi for 14 h. The reaction mixture isfiltered through sand and a membrane filter, then concentrated. Theresidue is dissolved in water (5 mL) and filtered. The filtrate ispurified by flash chromatography through a CHP-20 reverse-phase column(eluant: water, followed by 2% MeOH in water) to give 260 mg (44% yield)of the title compound as a white powder (m.p. 125-127° C.). TLC(trifluoroacetic acid:water 1:9) R_(f) 0.3. IR (KBr): 3395 (br), 2940,1640, 1630, 1450, 1150, 1075, 1047, 1023 cm⁻¹. ¹ H NMR (D₂ O): δ 5.09(br s, 1H), 4.87 (br s, 1H), 3.98 (br s, 1H), 3.78-2.88 (m, 21H),2.60-1.00 (m, 40H), 0.91 (s, 3H), 0.82 (d, 3H, J=5.1 Hz), 0.66 (s, 3H).

Hence, the present invention also contemplates various compoundsselected from non-glycosylated, monoglycosylated, and bis(glycosylated)bile acid-poly(aminoalkylene) or aminoarylene conjugates, including, inparticular, 3α,12α-dihydroxy-7-deoxy-5α-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide (deoxycholic acid-spermineconjugate); 3α-hydroxy-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide (bis(glycosylated)cholicacid-spermine conjugate);3α-hydroxy-12α-(1'α-glucosyl)-7-deoxy-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide (12α-(O-glucosyl)deoxycholicacid-spermine conjugate);3α-hydroxy-7α-(1'α-glucosyl)-12-deoxy-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide (7α-(O-glucosyl)chenodeoxycholicacid-spermine conjugate); 3α,7α,12α-trihydroxy-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide;3α,12α-dihydroxy-7α-deoxy-5β-cholan-24-oic acid,N-(12-aminododecyl)amide (deoxycholic acid-1,12-diaminododecaneconjugate); 3α,12α-dihydroxy-7-deoxy-5β-cholan-24-oic acid,N-(12-aminododecyl)amide;3α-hydroxy-7α,12α-di(2',3',4',6'-tetra-O-benzyl-1'.alpha.-glucosyl)-5β-cholan-24-oicacid, N-(4,9-diaza-12-aminododecyl)amide;3α,12α-dihydroxy-7-deoxy-5β-cholan-24-oic acid,N-(3,6,9-triaza-11-aminoundecyl)amide;3α,12α-dihydroxy-7-deoxy-5β-cholan-24-oic acid, N-(3,6,9,12-tetraaza-14-aminotetradecyl)amide;3α,7α-dihydroxy-12-deoxy-5-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide; 3β- and3α-amino-7α,12α-di(1'α-glucosyl)-5β-cholan-24-oic acid,N-(4,9-diaza-12-aminododecyl)amide; 3β- and3α-amino-7α,12α-di(2',3',4',6'-tetra-O-benzyl-1'α-glucosyl)-5β-cholan-24-oicacid, N-(4,9-diaza-12-aminododecyl)amide, intermediates in theirsyntheses described herein, and their pharmaceutically acceptable salts.

15. Biological Activity-Determination of MIC Against Bacteria

The results shown in Table 1 demonstrate that the compounds of thepresent invention exhibit activity useful in the treatment or preventionof infections.

The biological activity of the present compounds is demonstrated asfollows. To demonstrate their anti-infective properties, the minimuminhibitory concentration (MIC) for many of the novel compounds isobtained against a variety of antibiotic indicator strains of bacteria.Antibiotic indicator strains Escherichia coli strain 25922, Enterococcusfaecalis 29212, Pseudomonas aeruginosa 27853, and Staphylococcus aureus29213 are obtained from the American Type Tissue Culture Collection(ATCC) in Rockville, Md. The cystic fibrosis isolate, Pseudomonasaeruginosa 39324, is also obtained from ATCC. Bacteria are routinelycultivated in cation-supplemented Mueller-Hinton broth (CAMHB) or agarat 37° C.

The minimum inhibitory concentration (MIC) of glycosylated andnon-glycosylated steroidal polyamines for antibiotic indicator strainsis determined by dissolving the test compounds in deionized water to afinal concentration of 1 mg per mL. Those compounds that are poorlysoluble in water are dissolved in acetic acid, dried in a stream ofnitrogen gas, and dissolved in deionized water to a final concentrationof 1 mg per mL. These solutions are sterilized by filtration through0.22 micron syringe filters.

Stock solutions of individual compounds are serially diluted (two-fold)in sterile CAMHB in 96-well tissue culture dishes (Falcon) andinoculated with antibiotic indicator strains that are prepared asdescribed below. All compounds are tested in duplicate at concentrationsthat ranged from 1.56 to 200 μg per mL.

Antibiotic indicator strains are grown in 5 mL of CAMHB for 3-4 h at 37°C. with shaking (200 rpm) on a New Brunswick rotary shaker. Bacteria areadjusted to a turbidity that matched a 0.5 McFarland standard (ca. 10⁸CFU per mL) in sterile 0.85% saline. These bacterial suspensions arediluted 1:20 in sterile 0.85% saline and 10 μL (ca. 5×10⁵ CFU) of eachsuspension is used to inoculate individual wells of a 96 well plate thatcontained different concentrations of the test compounds. Followinginoculation, the plates are sealed with plastic tape, incubated for 24 hat 37° C. and visually inspected for bacterial growth. CAMHB inoculatedwith each of the antibiotic test strains and uninoculated CAMBH pluseach of the test compounds served as positive and negative controls. TheMIC is defined as the lowest concentration of a compound that completelyinhibited visual evidence of bacterial growth.

                                      TABLE 1                                     __________________________________________________________________________    Anti-Infective Properties Of Compounds                                         ##STR3##                                                                            MIC (μg/mL)                                                                                     E.  P.    E.  S.  P.                              Cmpd                        coli                                                                              aeurginosa                                                                          faecalis                                                                          aureus                                                                            aeruginosa                      No.    R.sup.1                                                                           R.sup.2                                                                            R.sup.3                                                                             Side Chain                                                                          25922                                                                             27853 29212                                                                             29213                                                                             39324                           __________________________________________________________________________    A      α-OH                                                                        H    OH    spermine                                                                            12.5                                                                              3.12  12.5                                                                              6.25                                                                              25                              B      α-OH                                                                        OH   OH    spermine                                                                            200 100-200                                                                             200 50  100                             C      α-OH                                                                        α-D-Glc                                                                      α-D-Glc                                                                       spermine                                                                            200 200   >200                                                                              200 --                              D      α-OH                                                                        H    α-D-Gln                                                                       spermine                                                                            100 100-200                                                                             200 50-100                                                                            --                              E      α-OH                                                                        α-D-Glc                                                                      H     spermine                                                                            12.5                                                                              12.5  25  25  12.5                            F      α-OH                                                                        H    OH    pentamine                                                                           100 100   100 25  100                             G      α-OH                                                                        H    OH    hexamine                                                                            100 50    100 25  50-100                          H      α-OH                                                                        OH   H     spermine                                                                            25  12.5  3.12                                                                              6.25                                                                              50-100                          I      α-OH                                                                        OH   H     pentamine                                                                           25  50    25  12.5                                                                              50                              J      α-OH                                                                        OH   OH    pentamine                                                                           75  75    18.7                                                                              37.5                                                                              37.5                            K      α-OH                                                                        OH   OH    hexamine                                                                            75  75    18.7                                                                              37.5                                                                              75                              L      α-OH                                                                        α-D-Glc                                                                      α-D-Glc                                                                       hexamine                                                                            >200                                                                              >200  >200                                                                              >200                                                                              >150                            M      α-OH                                                                        α-D-Glc                                                                      α-D-Glc                                                                       pentamine                                                                           >200                                                                              >200  >200                                                                              >200                                                                              >150                            N      α-OH                                                                        H    H     hexamine                                                                            >200                                                                              200   200 50  200                             pentaethylene-                                                                       --  --   --    --    >200                                                                              >200  >200                                                                              >200                                                                              >150                            hexamine                                                                      spermine                                                                             --  --   --    --    >200                                                                              >200  >200                                                                              >200                                                                              >150                            tetraethylene-                                                                       --  --   --    --    >200                                                                              >200  >200                                                                              >200                                                                              >150                            pentamine                                                                     cholic acid                                                                          --  --   --    --    >200                                                                              >200  >200                                                                              >200                                                                              >200                            sodium salt                                                                   deoxycholic                                                                          --  --   --    --    >200                                                                              >200  >200                                                                              >200                                                                              >200                            acid sodium                                                                   salt                                                                          __________________________________________________________________________

The nomenclature of compounds A to N has been provided earlier in thissepcification.

16. Augmentation Assay

The effect of the present compounds on the MIC of erythromycin,gentamicin and vancomycin for antibiotic indicator strains isdetermined, as described below.

Stock solutions 1-2 mg/mL of test compounds are prepared in distilledwater, filter-sterilized through a 0.22 μm filter and diluted in CAMHBto the desired starting concentration. Aliquots of each test compound(0.1 mL), at 2× the final concentration, are added to the first rows of96 well plates. The remaining wells of these plates are charged with 0.1mL aliquots of each test compound at the desired final concentration.Subsequently, 0.1 mL of solutions of erythromycin, gentamicin, orvancomycin, prepared in CAMHB, are added to the first rows of theseplates and serailly diluted.

The wells are inoculated in duplicate with each of four antibioticindicator strains prepared as described previously above. The finalconcentrations of antibiotics tested in these experiments range from0.185-250 μg per mL. Plates containing each of the antibiotics seriallydiluted in CAMHB alone are also inoculated with antibiotic indicatorstrains to determine the MIC of erythromycin, gentamicin and vancomycinin the absence of the compounds of the invention. The plates are sealedwith plastic tape, incubated in for 24 h at 37° C., visually inspectedfor bacterial growth. The MIC of erythromycin, gentamicin and vancomycinfor each of the indicator strains in the presence or absence of testcompound is subsequently determined. The results observed are listed inTable 2 below.

                  TABLE 2                                                         ______________________________________                                        Augmentation Of The Antibacterial Activity                                    Of Erythromycin                                                                      MIC (μg/mL)                                                         Erthromycin          P. aeruginosa                                                                             P. aeruginosa                                plus:    E. coli 25922                                                                             27853       39324                                        ______________________________________                                        no compound                                                                            125         >250        >250                                         compound B.sup.a                                                                       <0.19       3.12        3.12                                         compound D                                                                             0.39        0.39        6.25                                         compound H                                                                             <0.39       3.12        6.25                                         compound J                                                                             0.39        0.78        12.5                                         compound N                                                                             0.78        6.25        25                                           ______________________________________                                         .sup.a All compounds tested at 25 μg/mL, except compound H, which is       tested at 6.25 μg/mL. Bacteria grow in CAMHB supplemented with each        test compound at the indicated concentrations.                           

FIGS. 8A, 8B, 9A, 9B, 10A and 10B present selected results of theabove-described augmentation experiments in the form of histograms. Asshown in these figures, significant reductions in the MIC (μg/mL) oferythromycin are obtained with the co-administration of thisconventional anti-infective agent with selected compounds of theformula. Comparable results are shown or expected for the othercompounds of the formula.

The purpose of the above description and examples is to illustrate someembodiments of the present invention without implying any limitation. Itwill be apparent to those of skill in the art that various modificationsand variations may be made to the composition and method of the presentinvention without departing from the spirit or scope of the invention.All patents and publications cited herein are incorporated by referencein their entireties.

What is claimed is:
 1. A method of enhancing the anti-infective activityof an anti-infective agent comprising administering an effective dose ofan anti-infective agent in the presence of a compound of the formula (I)##STR4## in which; R₁ can be an H, OH, OR₅, NH₂, NHR₆ or NR₆ R₇ ;R₂ andR₃ may be the same or different and can be a H, OH or OR₅ ; R₄ can beCONH₂, CONHR₆, CONR₆ R₇, CH₂ NH₂, CH₂ NHR₆, CH₂ NR₆ R₇, CO₂ --Y--NH₂,CO₂ --Y--NHR₆, or CO₂ --Y--NR₆ R₇ ; R₅ is a protected or unprotectedglycosyl moiety comprising 1 -10 monosaccharide units in which theglycosidic linkage at the anomeric carbon atom of each monosaccharideunit is independently alpha or beta; NH₂, NHR₆, and NR₆ R₇ represent anunsubstituted amino group, a monosubstituted amino group, and adisubstituted amino group, respectively, in which R₆ and R₇ may be thesame or different and represent a hydrocarbon group comprising 1-15carbon atoms substituted with one or more unsubstituted, monosubstitutedor disubstituted amino groups; Y represents a linear or branchedalkylene group comprising 1-10 carbon atoms; n is an integer from 0-10;or its pharmaceutically acceptable salts; provided that if R₁ is OH, R₂is OH and R₃ is H, or OH; or if R₁ is OH, R₂ is H and R₃ is OH; then R₄cannot be a CONH₂, CONHCH₂ CH₂ N (C₂ H₅)₂, CON (CH₂ CH₂)₂ N--CH₃, CH₂NH₂ CH₂ NHCH₂ CH₂ N (C₂ H₅)₂, or CH₂ N (CH₂ CH₂)₂ N--CH₃ group.
 2. Themethod of claim 1 in which said anti-infective agent is selected fromthe group consisting of amikacin, bacitracin, candicidin, capreomycin,cephalosporins (cefazolin, cephaloglycine, cephaloridine, cephalothin,cephapirin sodium, cephradine), chloramphenicol, colistin (polymyxin),cycloserine, dactinomycin, erythromycin, fusidic acid, gentamicin,gramicidin, kanamycin, lincomycins (clindamycin, lincomycin), neomycin,oleandomycins (oleandomycin, troleandomycin), paromomycin, penicillins(amoxicillin, ampicillin, carbenicillin, carbenicillin, indanyl ester,cloxacillin, dicloxacillin, hetacillin, methacillin, nafcillin,oxacillin, penicillin G (benzylpenicillin), penicillin V(phenoxymetholpenicillin), phenethicillin), rifampin, spectinomycin,staphylomycin, streptomycins (dihydrostreptomycin, streptomycin),tetracyclines (chlortetracycline, demeclocycline, deoxycycline,methacycline, minocycline, oxytetracycline, tetracycline), tyrothricin,vancomycin, and viomycin.
 3. The method of claim 1 in which saidanti-infective agent is administered to a subject suffering from aninfection caused by an infectious microorganism.
 4. The method of claim1 in which the effective dose is about one-tenth or less the dosenecessary to achieve a comparable anti-infective activity when saidanti-infective agent is administered in the absence of the compound ofthe formula (I).
 5. The method of claim 3 in which said microorganismcauses skin and soft tissue infections, genitourinary-tract infections,gastrointestinal infections, gonorrhea, respiratory infections,meningitis, aspergillosis, cryptococcosis (torulosis), North Americanblastomycosis, systemic candidiasis, coccidioidomycosis, histoplasmosis,zygomycosis and subacute bacterial endocarditis.
 6. The method of claim3 in which said microorganism is selected from the group consisting ofalpha-streptococci, beta-streptococci, Diplococcus pneumoniae,Staphylococcus species, Bacillus anthracis, clostridia spp.,Corynebacterium xerose, Haemophilus ducreyi, Haemophilus influenzae,Escherichia coli, Klebsiella-Enterococcus species, Neisseria species,Proteus mirabilis, Salmonella typhosa, Pseudomonas aeruginosa,Histoplasma capsulatum, Coccidioides immitis, Candida species,Blastomyces dermatitidis, Rhondototorula, Cryptococcus neoformans,Sporothrix schenckii, Mucor mucedo and Aspergillus fumigatus.
 7. Themethod of claim 3 which further comprises contacting or co-contactingthe microorganism with an effective amount of one or more anti-infectiveagents other than a compound of the formula (I).